KR20230028589A - Novel cyclic dinucleotide derivative and antibody-drug conjugate thereof - Google Patents

Novel cyclic dinucleotide derivative and antibody-drug conjugate thereof Download PDF

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KR20230028589A
KR20230028589A KR1020237005621A KR20237005621A KR20230028589A KR 20230028589 A KR20230028589 A KR 20230028589A KR 1020237005621 A KR1020237005621 A KR 1020237005621A KR 20237005621 A KR20237005621 A KR 20237005621A KR 20230028589 A KR20230028589 A KR 20230028589A
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antibody
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drug conjugate
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KR102567591B1 (en
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도시후미 츠다
도시키 다부치
히데아키 와타나베
히로유키 고바야시
마사유키 이시자키
교코 하라
데이지 와다
마사미 아라이
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다이이찌 산쿄 가부시키가이샤
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Abstract

(과제) STING 아고니스트 활성을 갖는 신규 CDN 유도체 및 그 신규 CDN 유도체를 사용한 STING 아고니스트 활성이 관련하는 질환의 치료제 및/또는 치료법의 개발이 요망되고 있다. 또, 그 신규 CDN 유도체를 목적으로 하는 세포나 장기에 특이적으로 송달할 수 있는, STING 아고니스트 활성이 관련하는 질환의 치료제 및/또는 치료법의 개발이 요망되고 있다.
(해결 수단) 본 발명에 의해, 강한 STING 아고니스트 활성을 갖는 신규 CDN 유도체, 신규 CDN 유도체를 포함하는 항체 CDN 유도체 콘쥬게이트가 제공된다.(Problem) Development of a novel CDN derivative having STING agonist activity and a therapeutic agent and/or treatment for a disease associated with STING agonist activity using the novel CDN derivative is desired. Further, development of a therapeutic agent and/or therapeutic agent for a disease associated with STING agonist activity, which can specifically deliver the novel CDN derivative to a target cell or organ, is desired.
(Solution) According to the present invention, a novel CDN derivative having strong STING agonist activity and an antibody CDN derivative conjugate containing the novel CDN derivative are provided.

Description

신규 고리형 디뉴클레오티드 유도체 및 그 항체 약물 콘쥬게이트{NOVEL CYCLIC DINUCLEOTIDE DERIVATIVE AND ANTIBODY-DRUG CONJUGATE THEREOF}Novel cyclic dinucleotide derivative and its antibody drug conjugate {NOVEL CYCLIC DINUCLEOTIDE DERIVATIVE AND ANTIBODY-DRUG CONJUGATE THEREOF}

본 발명은, STING 아고니스트 활성을 갖는 신규 구조를 갖는 고리형 디뉴클레오티드 유도체, 그 신규 고리형 디뉴클레오티드 유도체와 표적 세포에 대한 항체를 링커를 개재하여 결합시킨 항체 약물 콘쥬게이트, 그 항체 약물 콘쥬게이트를 함유하는 의약 조성물 등에 관한 것이다.The present invention provides a cyclic dinucleotide derivative having a novel structure having STING agonist activity, an antibody drug conjugate obtained by linking the novel cyclic dinucleotide derivative with an antibody to a target cell via a linker, and the antibody drug conjugate. It relates to pharmaceutical compositions containing

STING (Stimulator of Interferon Genes) 은, 소포체에 국재하는 막 관통형의 어댑터 단백질이다 (비특허문헌 1). STING 은, 포유 동물에 있어서의 자연 면역 활성화의 중추 분자로서 기능하고 있고, 세균이나 바이러스와 같은 병원체의 진입에 대한 방어의 최전선을 담당하고 있다. STING 의 활성화는, 복수의 세포질 DNA 센서가, 외인성 및 내인성의 DNA 를 감지했을 때의 시그널에 의해 야기되는 것이 알려져 있다. 세포질 DNA 센서 중에서는, cGAS (Cyclic GMP-AMP Synthase) 가 중요한 DNA 센서라고 생각되고 있다. cGAS 가 DNA 를 감지하면 고리형 디뉴클레오티드 (2',3'-cGAMP) 가 산생되고, 이 2',3'-cGAMP 가 STING 에 직접 결합하여, STING 을 활성화시킨다 (비특허문헌 2). 활성화한 STING 은 골지체로 이동하고, 거기서 TBK1 (Tank-binding kinase 1) 의 자기 인산화를 재촉한다. 자기 인산화하여 활성화한 TBK-1 은, IRF3 (Interferon regulatory factor 3) 전사 경로 (비특허문헌 3) 및 NFκＢ 전사 경로 (비특허문헌 4) 의 양방을 활성화하고, 인터페론이나 사이토카인 (I 형 IFN (Interferon), IL-6 (Interleukin-6), TNF-α (Tumor Necrosis Factor-α)) 이라고 불리는 염증성 단백질의 산생을 증가시킨다. 이들 단백질은, 복잡한 캐스케이드를 통해서 병원체나 암 세포를 파괴하는 T 세포를 포함하는 획득 면역계를 시동시킨다.STING (Stimulator of Interferon Genes) is a transmembrane adapter protein localized in the endoplasmic reticulum (Non-Patent Document 1). STING functions as a central molecule for activation of natural immunity in mammals, and is responsible for the forefront of defense against the entry of pathogens such as bacteria and viruses. It is known that activation of STING is caused by a signal when a plurality of cytosolic DNA sensors detect exogenous and endogenous DNA. Among cytoplasmic DNA sensors, cGAS (Cyclic GMP-AMP Synthase) is considered to be an important DNA sensor. When cGAS senses DNA, cyclic dinucleotide (2',3'-cGAMP) is produced, and this 2',3'-cGAMP directly binds to STING to activate STING (Non-Patent Document 2). Activated STING moves to the Golgi apparatus, where it promotes autophosphorylation of TBK1 (Tank-binding kinase 1). TBK-1 activated by autophosphorylation activates both the IRF3 (Interferon regulatory factor 3) transcription pathway (Non-Patent Document 3) and the NFκB transcription pathway (Non-Patent Document 4), and interferons and cytokines (type I IFN ( Interferon), IL-6 (Interleukin-6), TNF-α (Tumor Necrosis Factor-α)) and increase the production of inflammatory proteins. These proteins initiate the acquiring immune system, which includes T cells, that destroy pathogens or cancer cells through a complex cascade.

최근 연구에 의해, STING 은 미생물에 대한 숙주 방어 뿐만 아니라, 항종양 면역을 촉진하는 것이 나타났다. 예를 들어, STING 결손 마우스에 면역원성 종양을 이식한 경우, 종양은, 야생형 마우스나 TRIF (Toll/Interleukin-1 (IL-1) receptor domain containing adaptor-inducing interferon-β) 결손 마우스보다 급속히 증식한다. 또, STING 결손 마우스에서는, TLR (Toll-like receptor), MyD88 (Myeloid differentiation primary response 88), MAVS (Mitochondrial antiviral-signaling protein) 결손 마우스와는 달리, 종양에 대한 자발적인 CD8＋ T 세포의 프라임도 소실하였다. 이것은 세포질 DNA 감지에 의해 시동하는 STING 경로가 종양 증식의 제어에 관여하고 있는 것을 시사하고 있다 (비특허문헌 5). 다른 연구에서는, STING 이 방사선 치료 (비특허문헌 6) 또는 항CD47 항체 요법 (비특허문헌 7) 의 항종양 효과에 필요한 것도 나타나 있다. 방사선 또는 항CD47 항체로 처치한 후의, 죽은 종양 세포 유래의 DNA 는, 수상 세포의 세포질로 이동하여 cGAS-STING 경로를 활성화한 후, IFN 산생을 유도하여 자연 면역과 획득 면역의 중개 역할을 한다. 이 연구는, STING 경로에 의해 활성화된 수상 세포를 개재하는 크로스 프라이밍이 종양에 대한 획득 면역을 일으키기 위해서 중요한 것을 시사하고 있다.Recent studies have shown that STING promotes antitumor immunity as well as host defense against microorganisms. For example, when immunogenic tumors are transplanted into STING-deficient mice, the tumors proliferate more rapidly than wild-type mice or TRIF (Toll/Interleukin-1 (IL-1) receptor domain containing adapter-inducing interferon-β)-deficient mice. . In addition, in STING-deficient mice, unlike TLR (Toll-like receptor), MyD88 (Myeloid differentiation primary response 88), and MAVS (mitochondrial antiviral-signaling protein) mice, spontaneous CD8 + T cell prime for tumors is also lost. did This suggests that the STING pathway triggered by cytoplasmic DNA sensing is involved in the control of tumor growth (Non-Patent Document 5). In another study, it has been shown that STING is required for the antitumor effect of radiation therapy (Non-Patent Document 6) or anti-CD47 antibody therapy (Non-Patent Document 7). DNA derived from dead tumor cells after treatment with radiation or anti-CD47 antibody migrates to the cytoplasm of dendritic cells, activates the cGAS-STING pathway, and then induces IFN production to play a mediating role between innate and acquired immunity. This study suggests that cross-priming via dendritic cells activated by the STING pathway is important for generating acquired immunity against tumors.

혈관 파괴제로서 알려져 있는 플라보노이드계 저분자 화합물 DMXAA 는, 매크로파지의 I 형 IFN 을 유도하기 때문에, 마우스 종양 모델에 있어서, 강력한 항종양 활성을 갖는 것이 나타났다 (비특허문헌 8). DMXAA 는 전(前) 임상에서의 우수한 항종양 효과 때문에, 비소세포 폐암의 면역요법약으로서 기대되고 있었지만, 인간 임상 시험에는 실패하였다 (비특허문헌 9). 최근 연구에 의해, DMXAA 는 마우스의 STING 에 대한 특이적인 아고니스트이고, 인간의 STING 에는 종 교차성이 없기 때문에, 결합할 수 없는 것이 밝혀졌다 (비특허문헌 10). DMXAA 는, 결과적으로 인간에서는 무효하였지만, 마우스 모델에 있어서의 연구에 의해, 저분자약이 STING 을 개재하여, 효과적으로 CD8＋ T 세포를 프라이밍하고, 항종양 면역을 증강할 수 있는 것이 시사되었다.Since DMXAA, a flavonoid-based low-molecular-weight compound known as a blood vessel disruptor, induces type I IFN of macrophages, it has been shown to have strong antitumor activity in a mouse tumor model (Non-Patent Document 8). DMXAA was expected as an immunotherapeutic drug for non-small cell lung cancer because of its excellent antitumor effect in preclinical studies, but failed in human clinical trials (Non-Patent Document 9). A recent study revealed that DMXAA is a specific agonist for mouse STING and cannot bind to human STING because it does not have cross-species compatibility (Non-Patent Document 10). Although DMXAA was ineffective in humans as a result, studies in a mouse model suggested that the small molecule drug could effectively prime CD8 + T cells via STING to enhance antitumor immunity.

다른 저분자 화합물로서, 고리형 디뉴클레오티드 (Cyclic dinucleotide, CDN) 는, 담암 (tumour-bearing) 마우스에 투여하면 STING 개재에 의한 항종양 면역 응답이 증강하여 종양 증식이 현저하게 저해되고, 마우스의 생존률을 개선하는 것이 나타났다 (비특허문헌 11). CDN 은, 세균 유래의 표준적인 2 개의 3'-5'인산 결합을 갖는 CDN (cyclic-di-GMP, cyclic-di-AMP, 3',3'-cGAMP) 과, 포유 동물의 cGAS 가 산생하는 비표준적인 2'-5'인산 결합을 갖는 혼합 결합형의 CDN (2',3'-cGAMP) 으로 분류된다. 최근 연구에 의해, 표준적인 CDN 보다 혼합 결합형의 CDN 쪽이 다양한 STING 을 보편적으로 활성화 가능한 것이 나타나 있다 (비특허문헌 12).As another low-molecular-weight compound, cyclic dinucleotide (CDN), when administered to tumor-bearing mice, enhances the anti-tumor immune response mediated by STING, significantly inhibits tumor growth, and improves the survival rate of mice. improvement has been shown (Non-Patent Document 11). CDN is a CDN (cyclic-di-GMP, cyclic-di-AMP, 3', 3'-cGAMP) having two standard 3'-5' phosphate bonds derived from bacteria and cGAS of mammals. It is classified as a mixed-linked CDN (2',3'-cGAMP) having a non-standard 2'-5' phosphate linkage. Recent studies have shown that a mixed binding type CDN can activate various STINGs more universally than a standard CDN (Non-Patent Document 12).

천연형의 CDN 은, 많은 핵산 분자와 같이 혈액 중의 핵산 분해 효소에 의해 신속하게 분해되기 때문에, 그대로의 형태로 투여할 수는 없다. 그 때문에, 생체내에서 STING 아고니스트 활성을 갖는 합성 저분자 화합물이 개발되고 있다 (예를 들어, 특허문헌 1 ∼ 26).Since native CDN is rapidly degraded by nucleases in the blood like many nucleic acid molecules, it cannot be administered in an intact form. Therefore, synthetic low-molecular compounds having STING agonist activity in vivo have been developed (for example, Patent Literatures 1 to 26).

현재, 항종양제로서 임상 시험이 진행되고 있는 STING 아고니스트의 MIW-815 (ADU-S100, ML RR-S2 CDA 또는 ML-RR-CDA·2Na＋ 라고 불리는 경우도 있다) 는, 종양 내에 직접 투여된다. STING 아고니스트를 종양에 직접 투여하는 방법으로는, 종양 내의 한정된 범위 밖에 약제를 투여할 수 없고, 또, 복수의 원격 전이 종양 전부에 직접 투여하는 것은 곤란하기 때문에, 치료 가능한 종양이 한정되어 버린다는 문제가 있다. 비특허문헌 13 에는, ML RR-S2 CDA 투여에 의해 항종양 효과가 나타난 것이 기재되지만, 종양내 투여 뿐이며, 전신 투여 (예를 들어, 정맥내 투여) 에 의한 항종양 효과는 개시되어 있지 않다. 비특허문헌 14 에는, STING 아고니스트의 SB11285 를 마우스 종양 모델에 정맥내 투여하면 항종양 효과가 나타난 것이 기재되지만, SB11285 가 구체적으로 어떠한 구조를 갖는 화합물인지는 밝혀져 있지 않다. 특허문헌 14 에는, 면역 자극 화합물과 항체 구축물과 링커를 함유하는 콘쥬게이트가 기재되어 있지만, 면역 자극 화합물로서 STING 아고니스트를 사용한 콘쥬게이트의 구체예는 기재되어 있지 않다. 특허문헌 26 에는, 특정한 구조를 갖는 CDN 과 항체를 링커를 개재하여 결합시킨 콘쥬게이트가 기재되어 있지만, 콘쥬게이트를 in vivo 에서 투여한 실시예의 기재가 없고, 콘쥬게이트의 항종양 효과는 확인되어 있지 않다.STING agonist MIW-815 (sometimes called ADU-S100, ML RR-S2 CDA or ML-RR-CDA 2Na + ), which is currently undergoing clinical trials as an antitumor agent, is administered directly into the tumor. do. In the method of directly administering the STING agonist to the tumor, the drug cannot be administered outside the limited range within the tumor, and it is difficult to directly administer to all of a plurality of distant metastatic tumors, so the tumors that can be treated are limited. there is a problem. Non-Patent Document 13 describes that an antitumor effect was exhibited by administration of ML RR-S2 CDA, but only intratumoral administration, and no antitumor effect by systemic administration (eg, intravenous administration) is disclosed. Non-Patent Document 14 describes that an antitumor effect was exhibited when SB11285, a STING agonist, was intravenously administered to a mouse tumor model, but the specific structure of SB11285 is unknown. Patent Document 14 describes a conjugate containing an immune stimulating compound, an antibody construct, and a linker, but does not disclose specific examples of the conjugate using a STING agonist as the immune stimulating compound. Patent Document 26 describes a conjugate in which a CDN having a specific structure and an antibody are linked via a linker, but there is no description of an example in which the conjugate is administered in vivo, and the antitumor effect of the conjugate has not been confirmed. not.

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STING 아고니스트 활성을 갖고, 인터페론이나 사이토카인 등의 염증성 단백질의 산생을 증가시켜 면역 세포를 부활화하는, 신규 골격을 갖는 CDN 유도체 및 그 신규 CDN 유도체를 사용한 STING 아고니스트 활성이 관련하는 질환, 예를 들어, 면역 부활화에 의한 치료가 가능한 질환 (예를 들어, 암) 의 치료제 및/또는 치료법의 개발이 요망되고 있다. 또, 전신 투여가 가능하고, 또한, 목적으로 하는 세포나 장기 (예를 들어, 종양 부위) 에 특이적으로 STING 아고니스트를 송달할 수 있는 그 신규 CDN 유도체와 표적 세포에 대한 항체를 링커를 개재하여 결합시킨 항체 약물 콘쥬게이트 및 그 항체 약물 콘쥬게이트를 사용한 STING 아고니스트 활성이 관련하는 질환, 예를 들어, 면역 부활화에 의한 치료가 가능한 질환 (예를 들어, 암) 의 치료제 및/또는 치료법의 개발이 요망되고 있다.CDN derivatives having STING agonist activity and activating immune cells by increasing the production of inflammatory proteins such as interferons and cytokines, and diseases associated with STING agonist activity using the novel CDN derivatives, e.g. For example, development of a therapeutic agent and/or therapeutic agent for a disease (for example, cancer) that can be treated by immune boosting has been desired. In addition, the novel CDN derivative capable of systemic administration and capable of delivering the STING agonist specifically to a target cell or organ (eg, tumor site) and an antibody against the target cell via a linker. Antibody drug conjugate conjugated thereto and a disease associated with STING agonist activity using the antibody drug conjugate, for example, a disease treatable by immunostimulation (eg, cancer), and/or treatment development is desired.

본 발명자들은, 상기의 과제를 해결하기 위해서, 축합 3 고리성 치환기를 갖는 것을 특징으로 하는 신규 CDN 유도체를 알아내어, 그 신규 CDN 유도체가, 강한 STING 아고니스트 활성을 갖고, 강한 항종양 활성을 나타내는 것을 알아냈다. 본 발명자들은 또한, 본원 발명의 신규 CDN 유도체와 항체를 링커를 개재하여 결합시킨 항체 약물 콘쥬게이트를 알아내어, 그 항체 약물 콘쥬게이트를 전신 투여하면, 항원이 발현하고 있는 종양에서 항종양 효과가 나타나는 것을 알아내어, 본 발명을 완성시켰다.In order to solve the above problems, the inventors of the present invention found a novel CDN derivative characterized by having a condensed tricyclic substituent, and the novel CDN derivative has strong STING agonist activity and exhibits strong antitumor activity. found out what The present inventors also found an antibody drug conjugate in which the novel CDN derivative of the present invention and an antibody were linked via a linker, and when the antibody drug conjugate was administered systemically, an antitumor effect was exhibited in a tumor expressing the antigen. found out, and completed the present invention.

즉 본원 발명은 이하에 관한 것이다. That is, the present invention relates to the following.

[1] 다음 식 (II) : [1] The following formula (II):

[화학식 1] [Formula 1]

Figure pat00001
Figure pat00001

(식 중, m1 은 1 내지 10 의 범위이고, (In the formula, m 1 is in the range of 1 to 10,

Ab 는, 항체 또는 그 항체의 기능성 단편을 나타내고, 그 항체의 당사슬은 리모델링 되어 있어도 되고, Ab represents an antibody or a functional fragment of the antibody, the sugar chain of the antibody may be remodeled,

L 은, Ab 와 D 를 연결하는 링커를 나타내고, L represents a linker linking Ab and D,

Ab 는 그 아미노산 잔기로부터 직접 L 에 결합하거나, Ab 의 당사슬 또는 리모델링 된 당사슬로부터 L 에 결합하고 있어도 되고, Ab may bind to L directly from its amino acid residue, or may bind to L from an Ab sugar chain or a remodeled sugar chain;

D 는, 다음 식 (I) : D is the following formula (I):

[화학식 2] [Formula 2]

Figure pat00002
Figure pat00002

(여기서, (here,

L 은, L1 또는 L2 에 포함되는 임의의 -NH2 또는 하이드록시기와 결합하고, L is bonded to any -NH 2 or hydroxyl group included in L 1 or L 2 ;

L1 은, 하이드록시기, -NH2, 2-하이드록시아세틸아미노메틸기 및 2-[(2-하이드록시아세틸)아미노]에틸기로 이루어지는 군에서 선택되는 1 내지 3 개의 기에 의해 임의의 위치에서 치환되어 있어도 되는, 다음 식L 1 is substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxyl group, -NH 2 , a 2-hydroxyacetylaminomethyl group and a 2-[(2-hydroxyacetyl)amino]ethyl group The following expression, which may be

[화학식 3] [Formula 3]

Figure pat00003
Figure pat00003

(여기서, (here,

R6 및 R6' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기, -NH2, C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기를 나타내고, R 6 and R 6' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 , a C1-C6 alkyl group, a C2-C6 alkenyl group or a C2-C6 alkynyl group;

R7 및 R7' 는, 각각 독립적으로, 수소 원자 또는 C1-C6 알킬기를 나타내고, 그 C1-C6 알킬기는, 할로겐 원자 및 옥소기로 이루어지는 군에서 선택되는 1 또는 2 개의 치환기에 의해 치환되어 있어도 되고, R 7 and R 7' each independently represent a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group may be substituted with 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group; ,

R8 및 R8' 는, 각각 독립적으로, 수소 원자 또는 할로겐 원자를 나타내고, R 8 and R 8' each independently represent a hydrogen atom or a halogen atom;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타내고, Z 4 represents -CH 2 -, -NH- or an oxygen atom;

Z5 는, 질소 원자 또는 -CH= 를 나타낸다) Z 5 represents a nitrogen atom or -CH=)

으로 이루어지는 군에서 선택되는 기를 나타내고, represents a group selected from the group consisting of

L2 는, 하기 (i) 또는 (ii) : L 2 is the following (i) or (ii):

(i) L 과 결합할 때, L2 는, -NHR', 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타내고, 여기서, R' 는, 수소 원자, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타내고, 그 C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기는, 1 내지 6 개의 할로겐 원자로 치환되어도 된다 ; 또는 (i) When combined with L, L 2 represents -NHR', a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group, wherein R' is a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alky represents a nyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group may be substituted with 1 to 6 halogen atoms; or

(ii) L 과 결합하지 않을 때, L2 는, 수소 원자 또는 할로겐 원자를 나타낸다, (ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom;

에서 선택되는 기를 나타내고, represents a group selected from

Q1 및 Q1' 는, 각각 독립적으로, 하이드록시기, 티올기 또는 보라노기 (BH3 -) 를 나타내고, Q 1 and Q 1' each independently represent a hydroxy group, a thiol group or a borano group (BH 3 - );

Q2 및 Q2' 는, 각각 독립적으로, 산소 원자 또는 황 원자를 나타내고, Q 2 and Q 2' each independently represent an oxygen atom or a sulfur atom;

X1 및 X2 는, 각각 독립적으로, 산소 원자, 황 원자 또는 -CH2- 를 나타내고, X 1 and X 2 each independently represent an oxygen atom, a sulfur atom or -CH 2 -;

Y1 및 Y2 는, 산소 원자 또는 -CH2- 를 나타내고, Y 1 and Y 2 represent an oxygen atom or -CH 2 -;

X3 및 X4 는, 하기 (iii) 또는 (iv) : X 3 and X 4 are the following (iii) or (iv):

(iii) Y1 이 산소 원자일 때, X3-X4 는, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (iii) when Y 1 is an oxygen atom, X 3 -X 4 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(iv) Y1 이 -CH2- 일 때, X3-X4 는, -O-CH2- 를 나타낸다, (iv) When Y 1 is -CH 2 -, X 3 -X 4 represents -O-CH 2 -;

에서 선택되는 기를 나타내고, represents a group selected from

X5 및 X6 은, 하기 (v) 또는 (vi) : X 5 and X 6 are (v) or (vi):

(v) Y2 가 산소 원자일 때, X5-X6 은, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (v) when Y 2 is an oxygen atom, X 5 -X 6 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(vi) Y2 가 -CH2- 일 때, X5-X6 은, -O-CH2- 를 나타낸다, (vi) When Y 2 is -CH 2 -, X 5 -X 6 represents -O-CH 2 -;

에서 선택되는 기를 나타내고, represents a group selected from

R1, R2 및 R3 은, 각각 독립적으로, 수소 원자, 할로겐 원자, -OR', -OC(=O)R', -N3, -NHR', -NR'R'' 또는 -NHC(=O)R' (여기서, R' 는, 앞에 정의한 바와 같고, R'' 는, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타낸다) 를 나타내고, R 1 , R 2 and R 3 are each independently a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N 3 , -NHR', -NR'R'' or -NHC (=O)R' (where R' is as defined above, and R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group) ,

W1 은, 질소 원자, 산소 원자, 황 원자 또는 -CH- 를 나타내고, W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom or -CH-;

W2 는, 질소 원자 또는 -CH= 를 나타내고, W 2 represents a nitrogen atom or -CH=;

R4 는, 수소 원자, 할로겐 원자 또는 -NH2 를 나타내고, R 4 represents a hydrogen atom, a halogen atom or -NH 2 ;

R5 는, 하기 (vii) 내지 (x) : R 5 is the following (vii) to (x):

(vii) W1 이 질소 원자일 때, R5 는 수소 원자, C1-C6 알킬기, 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타낸다 ; (vii) When W 1 is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group;

(viii) W1 이 산소 원자일 때, R5 는 존재하지 않고 ; (viii) when W 1 is an oxygen atom, R 5 is absent;

(ix) W1 이 황 원자일 때, R5 는 존재하지 않고 ; 또는 (ix) when W 1 is a sulfur atom, R 5 is absent; or

(x) W1 이 -CH- 일 때, R5 는 수소 원자, 할로겐 원자, 하이드록시기, -NH2 또는 C1-C6 알킬기를 나타낸다, (x) When W 1 is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 or a C1-C6 alkyl group;

에서 선택되는 기를 나타내고, represents a group selected from

Z1-Z2-Z3 은, 하나가 되어, -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타내고, X 는, 할로겐 원자를 나타낸다) 또는 하기 식 중 어느 것으로 나타내는 기Z 1 -Z 2 -Z 3 is one, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -R'''-, -CH=CH-CH 2 -, -CH= CX-CH 2 -, -CX=CH-CH 2 -, -CX=CX-CH 2 -, -C(=O)-CH 2 -CH 2 -, -CH 2 -CH 2 -C(=O) -, -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )- (where R''' is -O- or -CH 2 -CH 2 - and X represents a halogen atom) or a group represented by any of the following formulas

[화학식 4] [Formula 4]

Figure pat00004
Figure pat00004

(여기서, 아스테리스크는, W1 에 결합하고 있는 것을 나타내고, 파선은, =C- 의 탄소 원자에 결합하고 있는 것을 나타낸다) 에서 선택되는 기를 나타낸다) (Here, the asterisk indicates bonded to W 1 , and the broken line indicates bonded to a carbon atom of =C-).)

로 나타내는 화합물을 나타낸다) represents the compound represented by)

로 나타내는 항체 약물 콘쥬게이트 ; antibody drug conjugates represented by;

[2] W1 이 질소 원자인, [1] 에 기재된 항체 약물 콘쥬게이트 ; [2] The antibody drug conjugate according to [1], wherein W 1 is a nitrogen atom;

[3] W1 이 질소 원자이고, R5 가 수소 원자인, [2] 에 기재된 항체 약물 콘쥬게이트 ; [3] The antibody drug conjugate according to [2], wherein W 1 is a nitrogen atom and R 5 is a hydrogen atom;

[4] W1 이 산소 원자인, [1] 에 기재된 항체 약물 콘쥬게이트 ; [4] The antibody drug conjugate according to [1], wherein W 1 is an oxygen atom;

[5] W1 이 황 원자인, [1] 에 기재된 항체 약물 콘쥬게이트 ; [5] The antibody drug conjugate according to [1], wherein W 1 is a sulfur atom;

[6] W1 이 -CH- 인, [1] 에 기재된 항체 약물 콘쥬게이트 ; [6] the antibody drug conjugate described in [1], wherein W 1 is -CH-;

[7] W1 이 -CH- 이고, R5 가 수소 원자인, [6] 에 기재된 항체 약물 콘쥬게이트 ; [7] the antibody drug conjugate described in [6], wherein W 1 is -CH- and R 5 is a hydrogen atom;

[8] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-CH2- 또는 -CH=CH-CH2- 인, [1] ∼ [7] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [8] Z 1 , Z 2 and Z 3 are one, -CH 2 -CH 2 -CH 2 - or -CH=CH-CH 2 -, in any one of [1] to [7] antibody drug conjugates described;

[9] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- 인, [1] ∼ [7] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [9] Z 1 , Z 2 and Z 3 are one, -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )-, [1] to the antibody drug conjugate according to any one of [7];

[10] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-R'''- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타낸다) 인, [1] ∼ [7] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [10] Z 1 , Z 2 and Z 3 become one, -CH 2 -CH 2 -R'''- (where R''' is -O- or -CH 2 -CH 2 - shown), the antibody drug conjugate according to any one of [1] to [7];

[11] W2 가 -CH= 인, [1] ∼ [10] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [11] the antibody drug conjugate according to any one of [1] to [10], wherein W 2 is -CH=;

[12] W2 가 질소 원자인, [1] ∼ [10] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [12] the antibody drug conjugate according to any one of [1] to [10], wherein W 2 is a nitrogen atom;

[13] R4 가, 수소 원자를 나타내는, [1] ∼ [12] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [13] the antibody drug conjugate according to any one of [1] to [12], wherein R 4 represents a hydrogen atom;

[14] R4 가, 불소 원자를 나타내는, [1] ∼ [12] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [14] the antibody drug conjugate according to any one of [1] to [12], wherein R 4 represents a fluorine atom;

[15] L1 중의 R8 및 R8' 가, 각각 독립적으로, 수소 원자인, [1] ∼ [14] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [15] the antibody drug conjugate according to any one of [1] to [14], wherein R 8 and R 8' in L 1 are each independently a hydrogen atom;

[16] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[16] L 1 is a group selected from the group consisting of the following formula

[화학식 5][Formula 5]

Figure pat00005
Figure pat00005

(여기서, R9 및 R9' 는, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 9 and R 9' represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ,

R10 은, 하이드록시기, -NH2, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기를 나타내고, R 10 is a hydroxy group, -NH 2 , -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH, -CH 2 CH 2 NHC(=O)CH 2 OH, hydroxy represents a C1-C3 alkyl group or an amino C1-C3 alkyl group;

R11 및 R11' 는, 각각 독립적으로, 수소 원자, 불소 원자 혹은 메틸기를 나타내거나, 또는, R11 및 R11' 가 결합하여 시클로프로판을 나타내고, R 11 and R 11' each independently represent a hydrogen atom, a fluorine atom or a methyl group, or R 11 and R 11' combine to represent cyclopropane;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타낸다) Z 4 represents -CH 2 -, -NH- or an oxygen atom)

인, [1] ∼ [15] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; phosphorus, the antibody drug conjugate according to any one of [1] to [15];

[17] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[17] L 1 is a group selected from the group consisting of the following formula

[화학식 6][Formula 6]

Figure pat00006
Figure pat00006

(여기서, R13 및 R13' 는, 각각 독립적으로, 수소 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 13 and R 13' each independently represent a hydrogen atom, a hydroxyl group, or -NH 2 ,

R12 는, 하이드록시기, -NH2, -CH2OH, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH 또는 -CH2CH2NHC(=O)CH2OH 를 나타내고, R 12 is a hydroxy group, -NH 2 , -CH 2 OH, -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH or -CH 2 CH 2 NHC(=O)CH 2 OH,

Z4 는, 앞에 정의한 바와 같다) Z 4 is as defined above)

인, [1] ∼ [15] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; phosphorus, the antibody drug conjugate according to any one of [1] to [15];

[18] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[18] L 1 is a group selected from the group consisting of the following formula

[화학식 7] [Formula 7]

Figure pat00007
Figure pat00007

(여기서, R14 는, 수소 원자 또는 -NH2 를 나타내고, (Here, R 14 represents a hydrogen atom or -NH 2 ,

R15 는, 수소 원자 또는 -C(=O)CH2OH 를 나타내고, R 15 represents a hydrogen atom or -C(=O)CH 2 OH;

R16 은, 하이드록시기, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 또는 -CH2CH2NH2 를 나타낸다) R 16 represents a hydroxy group, -NH 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 NH 2 or -CH 2 CH 2 NH 2 )

인, [1] ∼ [15] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; phosphorus, the antibody drug conjugate according to any one of [1] to [15];

[19] L2 가, L 과 결합하고, -NH2, -CH2NH2 또는 -CH2OH 를 나타내는, [1] ∼ [18] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [19] The antibody drug conjugate according to any one of [1] to [18], wherein L 2 is bonded to L and represents -NH 2 , -CH 2 NH 2 or -CH 2 OH;

[20] L2 가, L 과 결합하지 않고, 수소 원자 또는 불소 원자를 나타내는, [1] ∼ [18] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [20] the antibody drug conjugate according to any one of [1] to [18], wherein L 2 is not bonded to L and represents a hydrogen atom or a fluorine atom;

[21] Q1 및 Q1' 가, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내는, [1] ∼ [20] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [21] the antibody drug conjugate according to any one of [1] to [20], wherein Q 1 and Q 1' each independently represent a hydroxy group or a thiol group;

[22] X1 및 X2 가, 산소 원자를 나타내는, [1] ∼ [21] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [22] the antibody drug conjugate according to any one of [1] to [21], wherein X 1 and X 2 represent oxygen atoms;

[23] Y1 및 Y2 가, 산소 원자를 나타내는, [1] ∼ [22] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [23] the antibody drug conjugate according to any one of [1] to [22], wherein Y 1 and Y 2 represent oxygen atoms;

[24] X3 및 X4 가, -CH2-O- 를 나타내는, [1] ∼ [23] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [24] the antibody drug conjugate according to any one of [1] to [23], wherein X 3 and X 4 represent -CH 2 -O-;

[25] X5 및 X6 이, -CH2-O- 를 나타내는, [1] ∼ [24] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [25] the antibody drug conjugate according to any one of [1] to [24], wherein X 5 and X 6 represent -CH 2 -O-;

[26] R1, R2 및 R3 이, 각각 독립적으로, 수소 원자, 하이드록시기 또는 불소 원자인, [1] ∼ [25] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 [26] The antibody drug conjugate according to any one of [1] to [25], wherein R 1 , R 2 and R 3 are each independently a hydrogen atom, a hydroxyl group or a fluorine atom.

[27] D 가, 이하의 2 식 : [27] D is, the following two formulas:

[화학식 8] [Formula 8]

Figure pat00008
Figure pat00008

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R17, R17', R18 및 R18' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, R 17 , R 17' , R 18 and R 18' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ;

W3 은, -NH-, 산소 원자, 황 원자 또는 -CH2- 를 나타내고, W 3 represents -NH-, an oxygen atom, a sulfur atom or -CH 2 -;

W4 는, -CH= 또는 질소 원자를 나타낸다) W 4 represents -CH= or a nitrogen atom)

중 어느 것으로 나타내는, [1] ∼ [26] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [1] to [26];

[28] D 가, 이하의 2 식 : [28] D is the following two formulas:

[화학식 9] [Formula 9]

Figure pat00009
Figure pat00009

(여기서, L1, Q1, Q1', Q2, Q2', R17, R17', R18 및 R18' 는, 앞에 정의한 바와 같다) (Where, L 1 , Q 1 , Q 1' , Q 2 , Q 2' , R 17 , R 17' , R 18 and R 18' are as defined above)

중 어느 것으로 나타내는, [27] 에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate described in [27], which is represented by any of the above;

[29] D 가, 이하의 8 식 : [29] D is, the following 8 expressions:

[화학식 10][Formula 10]

Figure pat00010
Figure pat00010

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R19, R19', R20 및 R20' 는, 각각 독립적으로, 수소 원자 또는 불소 원자를 나타낸다) R 19 , R 19' , R 20 and R 20' each independently represent a hydrogen atom or a fluorine atom)

중 어느 것으로 나타내는, [27] 또는 [28] 에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate described in [27] or [28], which is represented by any one;

[30] D 가, 이하의 4 식 : [30] D is, the following 4 equations:

[화학식 11] [Formula 11]

Figure pat00011
Figure pat00011

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [27] ∼ [29] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [27] to [29];

[31] D 가, 이하의 4 식 : [31] D is, the following 4 equations:

[화학식 12] [Formula 12]

Figure pat00012
Figure pat00012

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [27] ∼ [30] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [27] to [30];

[32] D 가, 이하의 4 식 : [32] D is, the following 4 equations:

[화학식 13] [Formula 13]

Figure pat00013
Figure pat00013

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [27] ∼ [30] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [27] to [30];

[33] D 가, 다음 식 : [33] D is, the following expression:

[화학식 14] [Formula 14]

Figure pat00014
Figure pat00014

(여기서, L1 은, 앞에 정의한 바와 같고,(Where L 1 is as defined above,

Q3 및 Q3' 는, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내고, Q 3 and Q 3' each independently represent a hydroxy group or a thiol group;

R21 및 R22 는, 각각 독립적으로, 하이드록시기 또는 불소 원자를 나타내고, R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom;

W5 는, -NH- 또는 황 원자를 나타낸다) W 5 represents -NH- or a sulfur atom)

으로 나타내는, [1] ∼ [26] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [1] to [26], represented by;

[34] D 가, 이하의 2 식 : [34] D is, the following two formulas:

[화학식 15] [Formula 15]

Figure pat00015
Figure pat00015

(여기서, L1, Q3 및 Q3', W5 는, 앞에 정의한 바와 같다) (Here, L 1 , Q 3 and Q 3 ' , W 5 are as defined above)

중 어느 것으로 나타내는, [33] 에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate described in [33], which is represented by any of the above;

[35] L1 이, 이하의 4 식 : [35] L 1 has the following 4 equations:

[화학식 16] [Formula 16]

Figure pat00016
Figure pat00016

중 어느 것으로 나타내는, [1] ∼ [34] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [1] to [34];

[36] L1 이, 이하의 4 식 : [36] L 1 has the following 4 equations:

[화학식 17] [Formula 17]

Figure pat00017
Figure pat00017

(식 중, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다) (In the formula, asterisk indicates bonding with L)

중 어느 것으로 나타내는, [1] ∼ [34] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [1] to [34];

[37] D 가, 이하의 4 식 : [37] D is, the following 4 equations:

[화학식 18] [Formula 18]

Figure pat00018
Figure pat00018

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타내고, Q3, Q3' 및 W5 는, 앞에 정의한 바와 같다) (Here, the asterisk indicates that it is bonded to L, and Q 3 , Q 3' and W 5 are as defined above)

중 어느 것으로 나타내는, [33], [34] 또는 [36] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [33], [34] and [36], which is represented by any of them;

[38] D 가, 이하의 4 식 : [38] D is, the following 4 equations:

[화학식 19] [Formula 19]

Figure pat00019
Figure pat00019

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다) (Here, the asterisk indicates that it is bonded to L)

중 어느 것으로 나타내는, [33], [34], [36] 또는 [37] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [33], [34], [36] or [37], which is represented by any of them;

[39] D 가, 이하의 3 식 : [39] D is, the following 3 formulas:

[화학식 20] [Formula 20]

Figure pat00020
Figure pat00020

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다) (Here, the asterisk indicates that it is bonded to L)

중 어느 것으로 나타내는, [33], [34], [36] 또는 [37] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [33], [34], [36] or [37], which is represented by any of them;

[40] D 가, 이하의 4 식 : [40] D is, the following 4 equations:

[화학식 21] [Formula 21]

Figure pat00021
Figure pat00021

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다) (Here, the asterisk indicates that it is bonded to L)

중 어느 것으로 나타내는, [33], [34], [36] 또는 [37] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [33], [34], [36] or [37], which is represented by any of them;

[41] 링커 L 이, -Lb-La-Lp-Lc-* 로 나타내고, [41] Linker L is represented by -Lb-La-Lp-Lc-*,

식 중, 아스테리스크는, 약물 D 와 결합하고 있는 것을 나타내고, In the formula, Asterisk represents binding to drug D,

Lp 는, 표적 세포에 있어서 절단 가능한 아미노산 서열로 이루어지는 링커를 나타내거나 또는 존재하지 않고, Lp represents or does not exist a linker consisting of an amino acid sequence cleavable in the target cell;

La 는, 이하의 군에서 선택되는 어느 1 개를 나타내고, La represents any one selected from the following groups,

-C(=O)-(CH2CH2)n2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-,

-C(=O)-(CH2CH2)n2-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -CH 2 -C(=O)-,

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-,

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -CH 2 -C(=O)-;

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2O)n3-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-,

-(CH2)n4-O-C(=O)-, 및, -(CH 2 )n 4 -OC(=O)-, and

-(CH2)n9-C(=O)- -(CH 2 )n 9 -C(=O)-

(여기서, n2 는 1 ∼ 3 의 정수를 나타내고, n3 은 1 ∼ 5 의 정수를 나타내고, n4 는 0 ∼ 2 의 정수를 나타내고, n9 는 2 ∼ 7 의 정수를 나타낸다), (Where n 2 represents an integer of 1 to 3, n 3 represents an integer of 1 to 5, n 4 represents an integer of 0 to 2, and n 9 represents an integer of 2 to 7),

Lb 는, La 와 Ab 의 당사슬 또는 리모델링 된 당사슬을 결합하는 스페이서 또는 La 와 Ab 의 시스테인 잔기를 결합하는 스페이서를 나타내고, 그리고, Lb represents a spacer that binds the sugar chains of La and Ab or the remodeled sugar chains or a spacer that binds the cysteine residues of La and Ab, and

Lc 는, -NH-CH2-, -페닐기-CH2-O(C=O)- 또는 -NH-헤테로아릴기-CH2-O(C=O)- 를 나타내거나 또는 존재하지 않는 Lc represents -NH-CH 2 -, -phenyl group-CH 2 -O(C=O)- or -NH-heteroaryl group-CH 2 -O(C=O)-, or is absent

[1] ∼ [40] 중 어느 하나에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [1] to [40];

[42] Lc 가 존재하지 않는, [41] 에 기재된 항체 약물 콘쥬게이트 ; [42] the antibody drug conjugate described in [41], wherein Lc is not present;

[43] Lc 가 -NH-CH2- 인, [41] 에 기재된 항체 약물 콘쥬게이트 ; [43] the antibody drug conjugate described in [41], wherein Lc is -NH-CH 2 -;

[44] Lp 가, 이하 : [44] Lp is, hereinafter:

-GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit- 및 -ICit- -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM -, -FM-, -GGLM-, -LM-, -GGICit- and -ICit-

로 이루어지는 군에서 선택되는 어느 하나를 나타내는, [41] ∼ [43] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [41] to [43], which is selected from the group consisting of;

[45] Lp 가, -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit- 또는 -FCit- 중 어느 1 개인, [44] 에 기재된 항체 약물 콘쥬게이트 ; [45] The antibody drug conjugate described in [44], wherein Lp is any one of -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, or -FCit- gate ;

[46] Lp 가, -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ- 또는 -GGPP- 중 어느 1 개인, [41] ∼ [43] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [46] Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ- or -GGPP-, the antibody drug conjugate according to any one of [41] to [43];

[47] Lp 가, -GGFG- 또는 -GGPI- 인, [46] 에 기재된 항체 약물 콘쥬게이트 ; [47] the antibody drug conjugate described in [46], wherein Lp is -GGFG- or -GGPI-;

[48] La 가, 이하 : [48] La, hereinafter:

-C(=O)-CH2CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, 및 -C(=0)-CH 2 CH 2 -C(=0)-NH-(CH 2 CH 2 O) 4 -CH 2 -C(=0)-, and

-(CH2)5-C(=O)- -(CH 2 ) 5 -C(=O)-

로 이루어지는 군에서 선택되는 어느 1 개를 나타내는, [41] ∼ [47] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; The antibody drug conjugate according to any one of [41] to [47], which is selected from the group consisting of;

[49] Lb 가, 다음 식[49] Lb is, the following equation

[화학식 22][Formula 22]

Figure pat00022
Figure pat00022

[화학식 23] [Formula 23]

Figure pat00023
Figure pat00023

또는,or,

[화학식 24] [Formula 24]

Figure pat00024
Figure pat00024

(상기에서 나타내는 Lb 의 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 당사슬 또는 리모델링 된 당사슬과 결합하고 있는 것을 나타낸다) 중 어느 것으로 나타내는, [41] ∼ [48] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; (In the structural formula of Lb shown above, the asterisk indicates that it is bonded to La, and the broken line indicates that it is bonded to the sugar chain or remodeled sugar chain of Ab), [41] to [48] the antibody drug conjugate according to any one of the above;

[50] Lb 가, -(숙신이미드-3-일-N)- 이고, [50] Lb is -(succinimide-3-yl-N)-,

여기서, -(숙신이미드-3-일-N)- 는, 이하의 구조식 : Here, -(succinimide-3-yl-N)- is the following structural formula:

[화학식 25] [Formula 25]

Figure pat00025
Figure pat00025

을 나타내고, represents,

여기서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 항체의 시스테인 잔기의 측사슬과 티오에테르를 형성하여 결합하고 있는 것을 나타내는, [41] ∼ [48] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; Here, the asterisk indicates binding to La, and the dashed line indicates binding by forming a thioether with the side chain of the cysteine residue of the antibody, the antibody drug according to any one of [41] to [48]. conjugate;

[51] 링커 L 이, -Lb-La-Lp-Lc-* 로 나타내고, [51] Linker L is represented by -Lb-La-Lp-Lc-*,

식 중, 아스테리스크는, 약물 D 와 결합하고 있는 것을 나타내고, In the formula, Asterisk represents binding to drug D,

Lp 가, -GGFG-, 또는 -GGPI- 이고, Lp is -GGFG- or -GGPI-;

La 가, -C(=O)-CH2CH2-C(=O)- 를 나타내고, La represents -C(=O)-CH 2 CH 2 -C(=O)-;

Lb 가, 다음 식Lb is, the following expression

[화학식 26] [Formula 26]

Figure pat00026
Figure pat00026

(상기에서 나타내는 Lb 의 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 당사슬 또는 리모델링 된 당사슬과 결합하고 있는 것을 나타낸다) 을 나타내고, (In the structural formula of Lb shown above, the asterisk indicates that it is bonded to La, and the broken line indicates that it is bonded to the sugar chain of Ab or the remodeled sugar chain),

Lc 가, -NH-CH2- 를 나타내는, [41], [46] ∼ [49] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; the antibody drug conjugate according to any one of [41], [46] to [49], wherein Lc represents -NH-CH 2 -;

[52] 항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 평균 약물 결합수가, 1 내지 10 개의 범위인, [1] ∼ [51] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [52] The antibody drug conjugate according to any one of [1] to [51], wherein the average number of drug bonds per antibody molecule in the antibody drug conjugate is in the range of 1 to 10;

[53] 항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 평균 약물 결합수가, 1 내지 5 개의 범위인, [52] 에 기재된 항체 약물 콘쥬게이트 ; [53] The antibody-drug conjugate according to [52], wherein the average number of drug bonds per antibody molecule in the antibody-drug conjugate is in the range of 1 to 5;

[54] 항체가, 항체의 Asn297 에 결합하는 당사슬 (N297 당사슬) 로부터 L 에 결합하고 있는, [1] ∼ [53] 중 어느 하나에 기재된 항체 약물 콘쥬게이트 ; [54] the antibody-drug conjugate according to any one of [1] to [53], wherein the antibody binds to L from the Asn297-binding sugar chain (N297 sugar chain) of the antibody;

[55] N297 당사슬이, 리모델링 된 당사슬인, [54] 에 기재된 항체 약물 콘쥬게이트 ; [55] the antibody drug conjugate described in [54], wherein the N297 sugar chain is a remodeled sugar chain;

[56] N297 당사슬이, N297-(Fuc)MSG1 또는 N297-(Fuc)SG 인, [54] 또는 [55] 에 기재된 항체 약물 콘쥬게이트 ; [56] the antibody drug conjugate according to [54] or [55], wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG;

[57] 항체가, 항HER2 항체, 항HER3 항체, 항DLL3 항체, 항FAP 항체, 항CDH11 항체, 항CDH6 항체, 항A33 항체, 항CanAg 항체, 항CD19 항체, 항CD20 항체, 항CD22 항체, 항CD30 항체, 항CD33 항체, 항CD56 항체, 항CD70 항체, 항CD98 항체, 항TROP2 항체, 항CEA 항체, 항Cripto 항체, 항EphA2 항체, 항G250 항체, 항MUC1 항체, 항GPNMB 항체, 항Integrin 항체, 항PSMA 항체, 항Tenascin-C 항체, 항SLC44A4 항체, 항Mesothelin 항체, 항ENPP3 항체, 항CD47 항체, 항EGFR 항체, 항GPR20 항체 또는 항DR5 항체인, [1] ∼ [56] 중 어느 한 항에 기재된 항체 약물 콘쥬게이트 ; [57] Antibody titer, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-CDH6 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, Anti-CD30 Antibody, Anti-CD33 Antibody, Anti-CD56 Antibody, Anti-CD70 Antibody, Anti-CD98 Antibody, Anti-TROP2 Antibody, Anti-CEA Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUC1 Antibody, Anti-GPNMB Antibody, Anti-Integrin An antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody or anti-DR5 antibody, any of [1] to [56] the antibody drug conjugate according to claim 1;

[58] 항체가, 항HER2 항체인, [57] 에 기재된 항체 약물 콘쥬게이트 ; [58] the antibody drug conjugate according to [57], wherein the antibody is an anti-HER2 antibody;

[59] 항체가, 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 2 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 또는, 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 3 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체인, [58] 에 기재된 항체 약물 콘쥬게이트 ; [59] The antibody is an antibody comprising a light chain composed of the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 2, or a light chain composed of the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: the antibody drug conjugate described in [58], which is an antibody comprising a heavy chain comprising the amino acid sequence described in 3;

[60] 항체가, 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 29 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 또는, 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 30 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체인, [58] 에 기재된 항체 약물 콘쥬게이트 ; [60] The antibody is an antibody comprising a light chain composed of the amino acid sequence shown in SEQ ID NO: 28 and a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 29, or a light chain composed of the amino acid sequence shown in SEQ ID NO: 28 and SEQ ID NO: the antibody drug conjugate described in [58], which is an antibody comprising a heavy chain comprising the amino acid sequence described in 30;

[61] 항체가, 서열 번호 31 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 32 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 서열 번호 33 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 34 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 또는, 서열 번호 35 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 36 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체인, [57] 에 기재된 항체 약물 콘쥬게이트 ; [61] The antibody comprises an antibody comprising a light chain composed of the amino acid sequence of SEQ ID NO: 31 and a heavy chain composed of the amino acid sequence of SEQ ID NO: 32, a light chain composed of the amino acid sequence of SEQ ID NO: 33, and SEQ ID NO: 34 The antibody drug according to [57], which is an antibody comprising a heavy chain comprising the amino acid sequence described or an antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 35 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 conjugate;

[62]다음 식 (Ia) : [62] The following formula (Ia):

[화학식 27] [Formula 27]

Figure pat00027
Figure pat00027

(여기서, (here,

L1 은, 하이드록시기, -NH2, 2-하이드록시아세틸아미노메틸기 및 2-[(2-하이드록시아세틸)아미노]에틸기로 이루어지는 군에서 선택되는 1 내지 3 개의 기에 의해 임의의 위치에서 치환되어 있어도 되는, 다음 식L 1 is substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxyl group, -NH 2 , a 2-hydroxyacetylaminomethyl group and a 2-[(2-hydroxyacetyl)amino]ethyl group The following expression, which may be

[화학식 28] [Formula 28]

Figure pat00028
Figure pat00028

(여기서, (here,

R6 및 R6' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기, -NH2, C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기를 나타내고, R 6 and R 6' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 , a C1-C6 alkyl group, a C2-C6 alkenyl group or a C2-C6 alkynyl group;

R7 및 R7' 는, 각각 독립적으로, 수소 원자 또는 C1-C6 알킬기를 나타내고, 그 C1-C6 알킬기는, 할로겐 원자 및 옥소기로 이루어지는 군에서 선택되는 1 또는 2 개의 치환기에 의해 치환되어 있어도 되고, R 7 and R 7' each independently represent a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group may be substituted with 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group; ,

R8 및 R8' 는, 각각 독립적으로, 수소 원자 또는 할로겐 원자를 나타내고, R 8 and R 8' each independently represent a hydrogen atom or a halogen atom;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타내고, Z 4 represents -CH 2 -, -NH- or an oxygen atom;

Z5 는, 질소 원자 또는 -CH= 를 나타낸다) Z 5 represents a nitrogen atom or -CH=)

으로 이루어지는 군에서 선택되는 기를 나타내고, represents a group selected from the group consisting of

L3 은, 수소 원자, 할로겐 원자, -NH2, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기를 나타내고, L 3 represents a hydrogen atom, a halogen atom, -NH 2 , a hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group;

Q1 및 Q1' 는, 각각 독립적으로, 하이드록시기, 티올기 또는 보라노기 (BH3 -) 를 나타내고, Q 1 and Q 1' each independently represent a hydroxy group, a thiol group or a borano group (BH 3 - );

Q2 및 Q2' 는, 각각 독립적으로, 산소 원자 또는 황 원자를 나타내고, Q 2 and Q 2' each independently represent an oxygen atom or a sulfur atom;

X1 및 X2 는, 각각 독립적으로, 산소 원자, 황 원자 또는 -CH2- 를 나타내고, X 1 and X 2 each independently represent an oxygen atom, a sulfur atom or -CH 2 -;

Y1 및 Y2 는, 산소 원자 또는 -CH2- 를 나타내고, Y 1 and Y 2 represent an oxygen atom or -CH 2 -;

X3 및 X4 는, 하기 (iii) 또는 (iv) : X 3 and X 4 are the following (iii) or (iv):

(iii) Y1 이 산소 원자일 때, X3-X4 는, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (iii) when Y 1 is an oxygen atom, X 3 -X 4 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(iv) Y1 이 -CH2- 일 때, X3-X4 는, -O-CH2- 를 나타낸다, (iv) When Y 1 is -CH 2 -, X 3 -X 4 represents -O-CH 2 -;

에서 선택되는 기를 나타내고, represents a group selected from

X5 및 X6 은, 하기 (v) 또는 (vi) : X 5 and X 6 are (v) or (vi):

(v) Y2 가 산소 원자일 때, X5-X6 은, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (v) when Y 2 is an oxygen atom, X 5 -X 6 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(vi) Y2 가 -CH2- 일 때, X5-X6 은, -O-CH2- 를 나타낸다, (vi) When Y 2 is -CH 2 -, X 5 -X 6 represents -O-CH 2 -;

에서 선택되는 기를 나타내고, represents a group selected from

R1, R2 및 R3 은, 각각 독립적으로, 수소 원자, 할로겐 원자, -OR', -OC(=O)R', -N3, -NHR', -NR'R'' 또는 -NHC(=O)R' (여기서, R' 는, 수소 원자, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타내고, 그 C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기는, 1 내지 6 개의 할로겐 원자로 치환되어도 된다, R'' 는, C1-6 알킬기, C2-6 알케닐기, C2-6 알키닐기 또는 C3-C6 시클로알킬기를 나타낸다) 를 나타내고, R 1 , R 2 and R 3 are each independently a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N 3 , -NHR', -NR'R'' or -NHC (=O)R' (wherein R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, the C1-C6 alkyl group, C2-C6 The alkenyl group or C2-C6 alkynyl group may be substituted with 1 to 6 halogen atoms, R'' represents a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group or a C3-C6 cycloalkyl group) represents,

W1 은, 질소 원자, 산소 원자, 황 원자 또는 -CH- 를 나타내고, W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom or -CH-;

W2 는, 질소 원자 또는 -CH= 를 나타내고, W 2 represents a nitrogen atom or -CH=;

R4 는, 수소 원자, 할로겐 원자 또는 -NH2 를 나타내고, R 4 represents a hydrogen atom, a halogen atom or -NH 2 ;

R5 는, 하기 (vii) 내지 (x) : R 5 is the following (vii) to (x):

(vii) W1 이 질소 원자일 때, R5 는 수소 원자, C1-C6 알킬기, 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타낸다 ; (vii) When W 1 is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group;

(viii) W1 이 산소 원자일 때, R5 는 존재하지 않고 ; (viii) when W 1 is an oxygen atom, R 5 is absent;

(ix) W1 이 황 원자일 때, R5 는 존재하지 않고 ; 또는 (ix) when W 1 is a sulfur atom, R 5 is absent; or

(x) W1 이 -CH- 일 때, R5 는 수소 원자, 할로겐 원자, 하이드록시기, -NH2 또는 C1-C6 알킬기를 나타낸다, (x) When W 1 is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 or a C1-C6 alkyl group;

에서 선택되는 기를 나타내고, represents a group selected from

Z1-Z2-Z3 은 하나가 되어, -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타내고, X 는, 할로겐 원자를 나타낸다) 또는 하기 식 중 어느 것으로 나타내는 기Z 1 -Z 2 -Z 3 become one, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -R'''-, -CH=CH-CH 2 -, -CH=CX -CH 2 -, -CX=CH-CH 2 -, -CX=CX-CH 2 -, -C(=O)-CH 2 -CH 2 -, -CH 2 -CH 2 -C(=O)- , -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )- (Where R''' represents -O- or -CH 2 -CH 2 - , X represents a halogen atom) or a group represented by any of the following formulas

[화학식 29] [Formula 29]

Figure pat00029
Figure pat00029

(여기서, 아스테리스크는, W1 에 결합하고 있는 것을 나타내고, 파선은, =C- 의 탄소 원자에 결합하고 있는 것을 나타낸다) 에서 선택되는 기를 나타낸다) (Here, the asterisk indicates bonded to W 1 , and the broken line indicates bonded to a carbon atom of =C-).)

로 나타내는 화합물 또는 그 약리상 허용되는 염 ; a compound represented by or a pharmacologically acceptable salt thereof;

[63] W1 이 질소 원자인, [62] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [63] the compound according to [62], wherein W 1 is a nitrogen atom, or a pharmacologically acceptable salt thereof;

[64] W1 이 질소 원자이고, R5 가 수소 원자인, [63] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [64] the compound according to [63] or a pharmacologically acceptable salt thereof, wherein W 1 is a nitrogen atom and R 5 is a hydrogen atom;

[65] W1 이 산소 원자인, [62] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [65] the compound according to [62], wherein W 1 is an oxygen atom, or a pharmacologically acceptable salt thereof;

[66] W1 이 황 원자인, [62] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [66] the compound according to [62], wherein W 1 is a sulfur atom, or a pharmacologically acceptable salt thereof;

[67] W1 이 -CH- 인, [62] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [67] the compound according to [62], wherein W 1 is -CH-, or a pharmacologically acceptable salt thereof;

[68] W1 이 -CH- 이고, R5 가 수소 원자인, [67] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; [68] the compound according to [67] or a pharmacologically acceptable salt thereof, wherein W 1 is -CH- and R 5 is a hydrogen atom;

[69] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-CH2- 또는 -CH=CH-CH2- 인, [62] ∼ [68] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [69] Z 1 , Z 2 and Z 3 are one, -CH 2 -CH 2 -CH 2 - or -CH=CH-CH 2 -, in any one of [62] to [68] the compounds described or their pharmacologically acceptable salts;

[70] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- 인, [62] ∼ [68] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [70] Z 1 , Z 2 and Z 3 are one, -CH 2 -CH (CH 3 ) -CH 2 - or -CH 2 -CH 2 -CH (CH 3 )-, [62] to the compound according to any one of [68] or a pharmacologically acceptable salt thereof;

[71] Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-R'''- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타낸다) 인, [62] ∼ [68] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [71] Z 1 , Z 2 and Z 3 become one, -CH 2 -CH 2 -R'''- (where R''' is -O- or -CH 2 -CH 2 - phosphorus, the compound according to any one of [62] to [68] or a pharmacologically acceptable salt thereof;

[72] W2 가 -CH= 인, [62] ∼ [71] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [72] the compound according to any one of [62] to [71], wherein W 2 is -CH= or a pharmacologically acceptable salt thereof;

[73] W2 가 질소 원자인, [62] ∼ [71] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [73] the compound according to any one of [62] to [71], wherein W 2 is a nitrogen atom, or a pharmacologically acceptable salt thereof;

[74] R4 가, 수소 원자를 나타내는, [62] ∼ [73] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [74] the compound according to any one of [62] to [73], wherein R 4 represents a hydrogen atom, or a pharmacologically acceptable salt thereof;

[75] R4 가, 불소 원자를 나타내는, [62] ∼ [73] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [75] the compound according to any one of [62] to [73], wherein R 4 represents a fluorine atom, or a pharmacologically acceptable salt thereof;

[76] L1 중의 R8 및 R8' 가, 각각 독립적으로, 수소 원자인, [62] ∼ [75] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [76] the compound according to any one of [62] to [75], or a pharmacologically acceptable salt thereof, wherein R 8 and R 8' in L 1 are each independently a hydrogen atom;

[77] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[77] L 1 is a group selected from the group consisting of the following formula

[화학식 30] [Formula 30]

Figure pat00030
Figure pat00030

(여기서, R9 및 R9' 는, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 9 and R 9' represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ,

R10 은, 하이드록시기, -NH2, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기를 나타내고, R 10 is a hydroxy group, -NH 2 , -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH, -CH 2 CH 2 NHC(=O)CH 2 OH, hydroxy represents a C1-C3 alkyl group or an amino C1-C3 alkyl group;

R11 및 R11' 는, 각각 독립적으로, 수소 원자, 불소 원자 혹은 메틸기를 나타내거나, 또는, R11 및 R11' 가 결합하여 시클로프로판을 나타내고, R 11 and R 11' each independently represent a hydrogen atom, a fluorine atom or a methyl group, or R 11 and R 11' combine to represent cyclopropane;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타낸다) Z 4 represents -CH 2 -, -NH- or an oxygen atom)

인, [62] ∼ [76] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; phosphorus, the compound according to any one of [62] to [76], or a pharmacologically acceptable salt thereof;

[78] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[78] L 1 is a group selected from the group consisting of the following formula

[화학식 31] [Formula 31]

Figure pat00031
Figure pat00031

(여기서, R13 및 R13' 는, 각각 독립적으로, 수소 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 13 and R 13' each independently represent a hydrogen atom, a hydroxyl group, or -NH 2 ,

R12 는, 하이드록시기, -NH2, -CH2OH, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH 또는 -CH2CH2NHC(=O)CH2OH 를 나타내고, R 12 is a hydroxy group, -NH 2 , -CH 2 OH, -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH or -CH 2 CH 2 NHC(=O)CH 2 OH,

Z4 는, 앞에 정의한 바와 같다) Z 4 is as defined above)

인, [62] ∼ [76] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; phosphorus, the compound according to any one of [62] to [76], or a pharmacologically acceptable salt thereof;

[79] L1 이, 다음 식으로 이루어지는 군에서 선택되는 기[79] L 1 is a group selected from the group consisting of the following formula

[화학식 32] [Formula 32]

Figure pat00032
Figure pat00032

(여기서, R14 는, 수소 원자 또는 -NH2 를 나타내고, (Here, R 14 represents a hydrogen atom or -NH 2 ,

R15 는, 수소 원자 또는 -C(=O)CH2OH 를 나타내고, R 15 represents a hydrogen atom or -C(=O)CH 2 OH;

R16 은, 하이드록시기, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 또는 -CH2CH2NH2 를 나타낸다) R 16 represents a hydroxy group, -NH 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 NH 2 or -CH 2 CH 2 NH 2 )

인, [62] ∼ [76] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; phosphorus, the compound according to any one of [62] to [76], or a pharmacologically acceptable salt thereof;

[80] L3 이, 수소 원자, 불소 원자, -NH2, -CH2OH 또는 -CH2NH2 를 나타내는, [62] ∼ [79] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [80] The compound according to any one of [62] to [79], wherein L 3 represents a hydrogen atom, a fluorine atom, -NH 2 , -CH 2 OH, or -CH 2 NH 2 , or a pharmacologically acceptable compound thereof. salt;

[81] Q1 및 Q1' 가, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내는, [62] ∼ [80] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [81] the compound according to any one of [62] to [80], or a pharmacologically acceptable salt thereof, wherein Q 1 and Q 1' each independently represent a hydroxy group or a thiol group;

[82] X1 및 X2 가, 산소 원자를 나타내는, [62] ∼ [81] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [82] the compound according to any one of [62] to [81], wherein X 1 and X 2 represent oxygen atoms, or a pharmacologically acceptable salt thereof;

[83] Y1 및 Y2 가, 산소 원자를 나타내는, [62] ∼ [82] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [83] the compound according to any one of [62] to [82], wherein Y 1 and Y 2 represent oxygen atoms, or a pharmacologically acceptable salt thereof;

[84] X3 및 X4 가, -CH2-O- 를 나타내는, [62] ∼ [83] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [84] the compound according to any one of [62] to [83], wherein X 3 and X 4 represent -CH 2 -O-, or a pharmacologically acceptable salt thereof;

[85] X5 및 X6 이, -CH2-O- 를 나타내는, [62] ∼ [84] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [85] the compound according to any one of [62] to [84], wherein X 5 and X 6 represent -CH 2 -O-, or a pharmacologically acceptable salt thereof;

[86] R1, R2 및 R3 이, 각각 독립적으로, 수소 원자, 하이드록시기 또는 불소 원자인, [62] ∼ [85] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; [86] the compound according to any one of [62] to [85], or a pharmacologically acceptable salt thereof, wherein R 1 , R 2 and R 3 are each independently a hydrogen atom, a hydroxyl group or a fluorine atom;

[87] 이하의 2 식 : [87] The following two equations:

[화학식 33] [Formula 33]

Figure pat00033
Figure pat00033

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R17, R17', R18 및 R18' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, R 17 , R 17' , R 18 and R 18' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ;

W3 은, -NH-, 산소 원자, 황 원자 또는 -CH2- 를 나타내고, W 3 represents -NH-, an oxygen atom, a sulfur atom or -CH 2 -;

W4 는, -CH= 또는 질소 원자를 나타낸다) W 4 represents -CH= or a nitrogen atom)

중 어느 것으로 나타내는, [62] ∼ [86] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [62] to [86], or a pharmacologically acceptable salt thereof;

[88] 이하의 2 식 : [88] The following two equations:

[화학식 34] [Formula 34]

Figure pat00034
Figure pat00034

(여기서, L1, Q1, Q1', Q2, Q2', R17, R17', R18 및 R18' 는, 앞에 정의한 바와 같다) (Where, L 1 , Q 1 , Q 1' , Q 2 , Q 2' , R 17 , R 17' , R 18 and R 18' are as defined above)

중 어느 것으로 나타내는, [87] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to [87], or a pharmacologically acceptable salt thereof, which is represented by any one;

[89] 이하의 8 식 : [89] Equation 8 below:

[화학식 35] [Formula 35]

Figure pat00035
Figure pat00035

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R19, R19', R20 및 R20' 는, 각각 독립적으로, 수소 원자 또는 불소 원자를 나타낸다) R 19 , R 19' , R 20 and R 20' each independently represent a hydrogen atom or a fluorine atom)

중 어느 것으로 나타내는, [87] 또는 [88] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; a compound according to [87] or [88], or a pharmacologically acceptable salt thereof;

[90] 이하의 4 식 : [90] 4 equations below:

[화학식 36] [Formula 36]

Figure pat00036
Figure pat00036

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [87] ∼ [89] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [87] to [89], or a pharmacologically acceptable salt thereof;

[91] 이하의 4 식 : [91] The following 4 equations:

[화학식 37] [Formula 37]

Figure pat00037
Figure pat00037

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [87] ∼ [90] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [87] to [90], or a pharmacologically acceptable salt thereof;

[92] 이하의 4 식 : [92] The following 4 equations:

[화학식 38] [Formula 38]

Figure pat00038
Figure pat00038

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타내는, [87] ∼ [90] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [87] to [90], or a pharmacologically acceptable salt thereof;

[93] 다음 식 : [93] The following expression:

[화학식 39] [Formula 39]

Figure pat00039
Figure pat00039

(여기서, L1 은, 앞에 정의한 바와 같고,(Where L 1 is as defined above,

Q3 및 Q3' 는, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내고, Q 3 and Q 3' each independently represent a hydroxy group or a thiol group;

R21 및 R22 는, 각각 독립적으로, 하이드록시기 또는 불소 원자를 나타내고, R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom;

W5 는, -NH- 또는 황 원자를 나타낸다) W 5 represents -NH- or a sulfur atom)

으로 나타내는, [62] ∼ [86] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [62] to [86], or a pharmacologically acceptable salt thereof, represented by;

[94] 이하의 2 식 : [94] The following two equations:

[화학식 40] [Formula 40]

Figure pat00040
Figure pat00040

(여기서, L1, Q3 및 Q3', W5 는, 앞에 정의한 바와 같다) (Here, L 1 , Q 3 and Q 3 ' , W 5 are as defined above)

중 어느 것으로 나타내는, [93] 에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to [93], or a pharmacologically acceptable salt thereof, which is represented by any one;

[95] L1 이, 이하의 : [95] L 1 has, or less:

[화학식 41] [Formula 41]

Figure pat00041
Figure pat00041

중 어느 것으로 나타내는, [62] ∼ [94] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [62] to [94], or a pharmacologically acceptable salt thereof;

[96] L1 이, 이하의 4 식 : [96] L 1 has the following 4 equations:

[화학식 42] [Formula 42]

Figure pat00042
Figure pat00042

중 어느 것으로 나타내는, [62] ∼ [94] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [62] to [94], or a pharmacologically acceptable salt thereof;

[97] D 가, 이하의 4 식 : [97] D has the following 4 equations:

[화학식 43] [Formula 43]

Figure pat00043
Figure pat00043

(여기서, Q3, Q3' 및 W5 는, 앞에 정의한 바와 같다) (Here, Q 3 , Q 3' and W 5 are as defined above)

중 어느 것으로 나타내는, [93], [94] 또는 [96] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [93], [94] or [96], or a pharmacologically acceptable salt thereof;

[98] D 가, 이하의 4 식 : [98] D has the following 4 equations:

[화학식 44] [Formula 44]

Figure pat00044
Figure pat00044

중 어느 것으로 나타내는, [93], [94], [96] 또는 [97] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [93], [94], [96] and [97], or a pharmacologically acceptable salt thereof;

[99] D 가, 이하의 3 식 : [99] D is, the following 3 formulas:

[화학식 45] [Formula 45]

Figure pat00045
Figure pat00045

중 어느 것으로 나타내는, [93], [94], [96] 또는 [97] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [93], [94], [96] and [97], or a pharmacologically acceptable salt thereof;

[100] D 가, 이하의 4 식 : [100] D is, the following 4 equations:

[화학식 46] [Formula 46]

Figure pat00046
Figure pat00046

중 어느 것으로 나타내는, [93], [94], [96] 또는 [97] 중 어느 한 항에 기재된 화합물 또는 그 약리상 허용되는 염 ; the compound according to any one of [93], [94], [96] and [97], or a pharmacologically acceptable salt thereof;

[101] [1] ∼ [61] 에 기재된 항체 약물 콘쥬게이트 및 [62] ∼ [100] 에 기재된 화합물 또는 그 약리상 허용되는 염으로 이루어지는 군에서 선택되는 어느 것을 함유하는, STING 아고니스트 ; [101] a STING agonist containing any one selected from the group consisting of the antibody drug conjugates described in [1] to [61] and the compounds described in [62] to [100] or pharmacologically acceptable salts thereof;

[102] [1] ∼ [61] 에 기재된 항체 약물 콘쥬게이트 및 [62] ∼ [100] 에 기재된 화합물 또는 그 약리상 허용되는 염으로 이루어지는 군에서 선택되는 어느 것을 함유하는, 의약 조성물 ; [102] A pharmaceutical composition containing any one selected from the group consisting of the antibody drug conjugate described in [1] to [61] and the compound described in [62] to [100] or a pharmacologically acceptable salt thereof;

[103] [1] ∼ [61] 에 기재된 항체 약물 콘쥬게이트 및 [62] ∼ [100] 에 기재된 화합물 또는 그 약리상 허용되는 염으로 이루어지는 군에서 선택되는 어느 것을 함유하는, 항종양제 ; [103] an antitumor agent containing any one selected from the group consisting of the antibody drug conjugate described in [1] to [61] and the compound described in [62] to [100] or a pharmacologically acceptable salt thereof;

[104] 종양이, 폐암, 신장암, 요로 상피암, 대장암, 전립선암, 다형 신경교아종, 난소암, 췌암, 유방암, 멜라노마, 간암, 방광암, 위암, 식도암, 자궁체암, 정소암, 자궁경암, 태반 융모암, 다형 신경교아종, 뇌종양, 두경부암, 갑상선암, 중피종, 소화관 간질 종양 (GIST), 담낭암, 담관암, 부신암, 유극세포암, 백혈병, 악성 림프종, 형질 세포종, 골수종 또는 육종인, [103] 에 기재된 항종양제 ; [104] Tumor, lung cancer, kidney cancer, urinary epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer [ 103];

[105] [1] ∼ [61] 에 기재된 항체 약물 콘쥬게이트, [62] ∼ [100] 에 기재된 화합물 또는 그 약리상 허용되는 염, [101] 에 기재된 STING 아고니스트, [102] 에 기재된 의약 조성물, 및 [103] 또는 [104] 에 기재된 항종양제로 이루어지는 군에서 선택되는 어느 것을 투여하는 것을 포함하는, 암의 치료 방법 ; [105] the antibody drug conjugate described in [1] to [61], the compound described in [62] to [100] or a pharmacologically acceptable salt thereof, the STING agonist described in [101], the drug described in [102] a method for treating cancer, comprising administering any one selected from the group consisting of a composition and an antitumor agent according to [103] or [104];

[106] 암이, 폐암, 신장암, 요로 상피암, 대장암, 전립선암, 다형 신경교아종, 난소암, 췌암, 유방암, 멜라노마, 간암, 방광암, 위암, 식도암, 자궁체암, 정소암, 자궁경암, 태반 융모암, 다형 신경교아종, 뇌종양, 두경부암, 갑상선암, 중피종, 소화관 간질 종양 (GIST), 담낭암, 담관암, 부신암, 유극세포암, 백혈병, 악성 림프종, 형질 세포종, 골수종 또는 육종인, [105] 에 기재된 방법, [106] Cancer, lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer [ 105],

에 관한 것이다.It is about.

본 발명에 의해, 신규 CDN 유도체가 제공된다. 본 발명의 신규 CDN 유도체는 강한 STING 아고니스트 활성을 갖고, 높은 항종양 활성을 나타낸다. 또, 본 발명에 의해, 전신 투여할 수 있고, 또한, 항원이 발현하고 있는 종양에서 항종양 효과를 나타내는, 신규 항체 CDN 유도체 콘쥬게이트가 제공된다.According to the present invention, novel CDN derivatives are provided. The novel CDN derivatives of the present invention have strong STING agonist activity and exhibit high antitumor activity. Moreover, according to the present invention, a novel antibody CDN derivative conjugate that can be administered systemically and exhibits an antitumor effect in a tumor expressing an antigen is provided.

도 1 은, 본 발명의 약물 콘쥬게이트체 ((II) 의 분자) 인, SG 형 당사슬 리모델링 항체로부터 얻어진 약물 콘쥬게이트체 (도 1 의 A 의 (II) 의 분자) 및, MSG 형 당사슬 리모델링 항체로부터 얻어진 약물 콘쥬게이트체 (도 1 의 B 의 (II) 의 분자) 를 모식적으로 나타낸 것이다. (a) 는 약물 D, (b) 는 링커 L, (c) 는 PEG 링커 (L(PEG)), (d) 는 N297 당사슬 (여기서, 백색 원은 NeuAc(Sia), 백색 육각형은 Man, 흑색 육각형은 GlcNAc, 백색 마름모꼴은 Gal, 및, 백색 역삼각형은 Fuc) 을 각각 나타낸다. 백색 오각형은, 링커 L 유래의 알킨과 PEG 링커 유래의 아지드기가 반응하여 생성된 트리아졸 고리를 나타낸다. Y 자형은, 항체 Ab 를 나타낸다. PEG 링커는, 비환원 말단에 위치하는 시알산의 2 위치의 카르복실기와 아미드 결합을 개재하여 연결되어 있다. 이와 같은 표시 방법은, 특별히 언급이 없는 한, 본 명세서의 전체를 통해 적용된다.
도 2 는, 본 발명의 약물 콘쥬게이트체의 제조 중간체인, (Fucα1,6)GlcNAc-항체 (도 2 의 A 의 (III) 의 분자), SG 형 당사슬 리모델링 항체 (도 2 의 B 의 (IV) 의 분자), 및 MSG 형 당사슬 리모델링 항체 (도 2 의 C 의 (IV) 의 분자) 의 구조를 나타내는 모식도이다. 모든 도면에 있어서, Y 자형은 도 1 과 마찬가지로 항체 Ab 를 나타낸다. 도 2 의 A 에 있어서, (e) 는 Fuc 의 1 위치와, GlcNAc 의 6 위치에 있어서 α글리코시드 결합한 이당으로 이루어지는 N297 당사슬을 나타낸다. 도 2 의 B 및 C 에 있어서, (d) 는 도 1 과 동일한 N297 당사슬을 나타내고, (f) 는 아지드기를 갖는 PEG 링커이며, 말단에 링커 L 과의 결합에 제공되는 아지드기를 나타낸다. 아지드기를 갖는 PEG 링커의 결합 양식은, 도 1 의 PEG 링커와 동일하다.
도 3 은, 동물 세포에서 산생된 항체로부터 SG 형 당사슬 리모델링 항체 및 MSG 형 당사슬 리모델링 항체를 제조하는 공정의 모식도이다. 도면 중의 분자 (III) 및 (IV) 는, 도 2 와 마찬가지로, (Fucα1,6)GlcNAc-항체 및 SG 형 당사슬 리모델링 항체 또는 MSG 형 당사슬 리모델링 항체를 각각 나타낸다. (V) 의 분자는 동물 세포에서 산생된 항체이며, N297 당사슬이 불균일한 분자의 혼합물이다. 도 3A 는, (V) 의 불균일한 N297 당사슬을, EndoS 와 같은 가수분해 효소로 처리함으로써, 균일한 (Fucα1,6)GlcNAc-항체 (III) 이 제조되는 공정을 나타낸다. 도 3B 는, 항체 (III) 의 N297 당사슬의 GlcNAc 에 대하여, EndoS D233Q/Q303L 변이체와 같은 당전이 효소를 사용하여, SG 형 당사슬 도너 분자를 당사슬 전이시킴으로써, (IV) 의 SG 형 당사슬 리모델링 항체가 제조되는 공정을 나타낸다. 도 3C 는, 도 3B 와 마찬가지로, 항체 (III) 에 대하여, MSG 형 당사슬 도너 분자를 당사슬 전이시킴으로써 (IV) 의 MSG 형 당사슬 리모델링 항체가 제조되는 공정을 나타낸다. 여기서 사용되는 SG 형 당사슬 도너 분자 및 MSG 형 당사슬 도너 분자는, 각각의 비환원 말단의 시알산이 아지드기를 갖는 PEG 링커로 수식된 것이고, 제조되는 SG 형 N297 당사슬 리모델링 항체 및 MSG 형 N297 당사슬 리모델링 항체에 있어서도, 도 2 의 B 및 C 에서 도시한 바와 같이, 비환원 말단의 시알산이 동일한 수식을 받은 것이 된다.
도 4 는, 트라스투주맙의 경사슬의 아미노산 서열 (서열 번호 1) 및 중사슬의 아미노산 서열 (서열 번호 2) 을 나타낸다.
도 5 는, 개변 항HER2 항체의 경사슬의 아미노산 서열 (서열 번호 1) 및 중사슬의 아미노산 서열 (서열 번호 3) 을 나타낸다.
도 6 은, (a) 인간 STING 야생형의 아미노산 서열, (b) 인간 STING REF 변이형 (R232H) 의 아미노산 서열, 및, (c) 인간 STING HAQ 변이체 (R71H, G230A, R293Q) 의 아미노산 서열을 나타낸다.
도 7 은, CDN 유도체의 종양내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 사각선은 화합물 번호 6a 투여군, 백색 역삼각선은 화합물 번호 8b 투여군, 백색 동그라미선은 화합물 번호 9b 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 8 은, 항HER2 항체 - CDN 콘쥬게이트 (1) 및 항LPS 항체 - CDN 콘쥬게이트 (1) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은, 참고예 1 에서 제조한 개변 항HER2 항체에 실시예 8b 의 화합물을 콘쥬게이트시킨 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군, 흑색 삼각선은, 참고예 2 에서 제조한 개변 항LPS 항체에 동일하게 실시예 8b 의 화합물을 콘쥬게이트시킨 항LPS 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 9 는, 항HER2 항체 - CDN 콘쥬게이트 (2) 및 (3) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 사각선은 항HER2 항체 - CDN 콘쥬게이트 (2) 투여군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (3) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 10 은, 항HER2 항체 - CDN 콘쥬게이트 (19) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (19) 투여군을 나타낸다. 항HER2 항체 - CDN 콘쥬게이트 (19) 는 약물 링커가 시스테인 콘쥬게이션으로 항체에 결합되어 있다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 11 은, 항HER2 항체 - CDN 콘쥬게이트 (1) 및 (9) ∼ (12) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (9) 투여군, 백색 역삼각선은 항HER2 항체 - CDN 콘쥬게이트 (10) 투여군, 백색 마름모꼴선은 항HER2 항체 - CDN 콘쥬게이트 (11) 투여군, 백색 동그라미선은 항HER2 항체 - CDN 콘쥬게이트 (12) 투여군, 백색 사각선은 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 항HER2 항체 - CDN 콘쥬게이트 (9), (10), (11), (12), (1) 은 각각 상이한 링커로 실시예 8b 의 화합물이 콘쥬게이션되어 있다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 12 는, 항HER2 항체 2 - CDN 콘쥬게이트 (1), 항HER2 항체 2 및 화합물 번호 8b 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (1) 의 60 ㎍ 투여군, 흑색 역삼각선은 항HER2 항체 2 의 59 ㎍ 투여군, 흑색 동그라미선은 화합물 번호 8b 의 1.2 ㎍ 투여군을 나타낸다. 항HER2 항체 2 및 화합물 번호 8b 의 투여량은, 항HER2 항체 2 - CDN 콘쥬게이트 (1) 을 구성하는 각 성분의 당량이다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 13 은, (a) 항HER2 항체 2 - CDN 콘쥬게이트 (2) 및 (3) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. (b) 항HER2 항체 2 - CDN 콘쥬게이트 (4), (5), (7) 및 (8) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. (c) 항HER2 항체 2 - CDN 콘쥬게이트 (6) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 각 백색 심볼선은 평가한 각 항HER2 항체 2 - CDN 콘쥬게이트 (2) ∼ (8) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 14 는, 항HER2 항체 2 - CDN 콘쥬게이트 (9) 및 (10) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (9) 투여군, 백색 동그라미선은 항HER2 항체 2 - CDN 콘쥬게이트 (10) 투여군을 나타낸다. 항HER2 항체 2 - CDN 콘쥬게이트 (9), (10) 은 약물 평균 결합수가 약 2 인 MSG 형 당사슬 리모델링 항체를 사용한 항체 - CDN 콘쥬게이트이다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 15 는, 항EphA2 항체 및 항EphA2 항체 - CDN 콘쥬게이트 (1) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 동그라미선은 항EphA2 항체 투여군, 백색 삼각선은 항EphA2 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 16 은, 항CD33 항체 및 항CD33 항체 - CDN 콘쥬게이트 (1) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 동그라미선은 항CD33 항체 투여군, 백색 삼각선은 항CD33 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다.
도 17 은, 페르투주맙의 경사슬의 아미노산 서열 (서열 번호 28) 및 중사슬의 아미노산 서열 (서열 번호 29) 을 나타낸다.
도 18 은, 개변 항HER2 항체 2 의 경사슬의 아미노산 서열 (서열 번호 28) 및 중사슬의 아미노산 서열 (서열 번호 30) 을 나타낸다.
도 19 는, 항CD33 항체의 경사슬의 아미노산 서열 (서열 번호 31) 및 중사슬의 아미노산 서열 (서열 번호 32) 을 나타낸다.
도 20 은, 항EphA2 항체의 경사슬의 아미노산 서열 (서열 번호 33) 및 중사슬의 아미노산 서열 (서열 번호 34) 을 나타낸다.
도 21 은, 항CDH6 항체의 경사슬의 아미노산 서열 (서열 번호 35) 및 중사슬의 아미노산 서열 (서열 번호 36) 을 나타낸다.
도 22 는, 항HER2 항체 2 - CDN 콘쥬게이트 (11) 및 (12) 의 정맥내 투여에 의한 항종양 효과를 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (11) 투여군, 백색 동그라미선은 항HER2 항체 2 - CDN 콘쥬게이트 (12) 투여군을 나타낸다.Fig. 1 shows the drug conjugate (molecule of (II) in Fig. 1 A) obtained from the SG-type sugar chain remodeling antibody, which is the drug conjugate ((II) molecule) of the present invention, and the MSG-type sugar chain remodeling antibody It schematically shows the drug conjugate obtained from (the molecule of (II) in B in FIG. 1). (a) is drug D, (b) is linker L, (c) is PEG linker (L(PEG)), (d) is N297 sugar chain (where white circle is NeuAc(Sia), white hexagon is Man, black Hexagons represent GlcNAc, white diamonds represent Gal, and white inverted triangles represent Fuc), respectively. The white pentagon represents a triazole ring formed by the reaction of an alkyne derived from linker L with an azide group derived from a PEG linker. Y-shape represents antibody Ab. The PEG linker is linked to the 2-position carboxyl group of sialic acid located at the non-reducing end via an amide bond. Such a display method is applied throughout the present specification unless otherwise specified.
Figure 2 shows the production intermediates of the drug conjugate of the present invention, (Fucα1,6)GlcNAc-antibody (molecule of (III) in Figure 2A), SG-type sugar chain remodeling antibody ((IV of Figure 2B) ), and MSG-type sugar chain remodeling antibody (molecule of (IV) in C of FIG. 2). In all figures, Y-shape represents antibody Ab as in FIG. 1 . In Fig. 2A, (e) represents the N297 sugar chain composed of a disaccharide linked by α-glycosidic bonds at the 1-position of Fuc and the 6-position of GlcNAc. In B and C of FIG. 2, (d) represents the same N297 sugar chain as in FIG. 1, and (f) represents a PEG linker having an azide group, and an azide group provided for bonding with linker L at the terminal. The bonding mode of the PEG linker having an azide group is the same as that of the PEG linker in FIG. 1 .
Fig. 3 is a schematic diagram of a process for producing SG-type sugar chain remodeling antibodies and MSG-type sugar chain remodeling antibodies from antibodies produced in animal cells. Molecules (III) and (IV) in the figure represent (Fucα1,6)GlcNAc-antibodies and SG-type sugar chain remodeling antibodies or MSG-type sugar chain remodeling antibodies, respectively, as in FIG. 2 . The molecule of (V) is an antibody produced in an animal cell, and is a mixture of molecules having heterogeneous N297 sugar chains. Fig. 3A shows a process for producing a homogeneous (Fucα1,6)GlcNAc-antibody (III) by treating the heterogeneous N297 sugar chain of (V) with a hydrolase such as EndoS. Fig. 3B shows that the SG-type sugar chain remodeling antibody of (IV) is obtained by transferring an SG-type sugar chain donor molecule to GlcNAc of the N297 sugar chain of antibody (III) using a glycosyltransferase such as the EndoS D233Q/Q303L variant. Indicates the manufacturing process. Fig. 3C, similar to Fig. 3B, shows a process for preparing the MSG-type sugar chain remodeling antibody (IV) by transferring the MSG-type sugar chain donor molecule to the antibody (III). The SG-type sugar chain donor molecule and the MSG-type sugar chain donor molecule used herein are those in which the sialic acid at each non-reducing end is modified with a PEG linker having an azide group, and the produced SG-type N297 sugar chain remodeling antibody and MSG-type N297 sugar chain remodeling antibody , as shown in B and C of Fig. 2, the sialic acid at the non-reducing end is the same modified.
4 shows the amino acid sequence of the light chain (SEQ ID NO: 1) and the amino acid sequence of the heavy chain (SEQ ID NO: 2) of Trastuzumab.
Fig. 5 shows the light chain amino acid sequence (SEQ ID NO: 1) and heavy chain amino acid sequence (SEQ ID NO: 3) of the modified anti-HER2 antibody.
6 shows (a) the amino acid sequence of human STING wild type, (b) the amino acid sequence of human STING REF variant (R232H), and (c) the amino acid sequence of human STING HAQ variants (R71H, G230A, R293Q) .
Fig. 7 shows the antitumor effect of intratumoral administration of a CDN derivative. In the figure, the black square line indicates the vehicle group, the white square line indicates the Compound No. 6a administration group, the white inverted triangle indicates the Compound No. 8b administration group, and the open circle line indicates the Compound No. 9b administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 8 shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugate (1) and anti-LPS antibody-CDN conjugate (1). In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody-CDN conjugate (1) administration group in which the compound of Example 8b was conjugated to the modified anti-HER2 antibody prepared in Reference Example 1, and the black triangle indicates , Anti-LPS antibody-CDN conjugate (1) administration group in which the compound of Example 8b was conjugated to the modified anti-LPS antibody prepared in Reference Example 2 in the same way. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 9 shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugates (2) and (3). In the figure, the black square line indicates the vehicle group, the white square line indicates the anti-HER2 antibody-CDN conjugate (2) administration group, and the open triangle indicates the anti-HER2 antibody-CDN conjugate (3) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 10 shows the antitumor effect of intravenous administration of the anti-HER2 antibody-CDN conjugate (19). The black square line in the figure represents the vehicle group, and the open triangle represents the anti-HER2 antibody-CDN conjugate (19) administration group. In the anti-HER2 antibody-CDN conjugate (19), a drug linker is linked to the antibody by cysteine conjugate. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 11 shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugates (1) and (9) to (12). In the figure, the black square line is the vehicle group, the white triangle is the anti-HER2 antibody-CDN conjugate (9) administration group, the white inverted triangle is the anti-HER2 antibody-CDN conjugate (10) administration group, and the white diamond line is the anti-HER2 antibody-CDN The conjugate (11) administration group, the open circle line represents the anti-HER2 antibody-CDN conjugate (12) administration group, and the open square line represents the anti-HER2 antibody-CDN conjugate (1) administration group. In the anti-HER2 antibody-CDN conjugates (9), (10), (11), (12) and (1), the compound of Example 8b was conjugated with different linkers. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 12 shows the antitumor effect of intravenous administration of anti-HER2 antibody 2 - CDN conjugate (1), anti-HER2 antibody 2 and Compound No. 8b. In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody 2-CDN conjugate (1) 60 μg administration group, the black inverted triangle indicates the anti-HER2 antibody 2 59 μg administration group, and the black dotted line indicates Compound No. 8b. The 1.2 μg administration group is shown. The doses of anti-HER2 antibody 2 and Compound No. 8b are equivalents of each component constituting the anti-HER2 antibody 2-CDN conjugate (1). The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 13 shows (a) the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (2) and (3). (b) Intravenous administration of anti-HER2 antibody 2-CDN conjugates (4), (5), (7) and (8) shows antitumor effects. (c) Antitumor effect by intravenous administration of anti-HER2 antibody 2-CDN conjugate (6) is shown. The black square line in the figure represents the vehicle group, and each white symbol line represents the anti-HER2 antibody 2-CDN conjugate (2) to (8) administration groups evaluated. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 14 shows the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (9) and (10). In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody 2-CDN conjugate (9) administration group, and the open circle line indicates the anti-HER2 antibody 2-CDN conjugate (10) administration group. Anti-HER2 antibody 2-CDN conjugates (9) and (10) are antibody-CDN conjugates using MSG-type sugar chain remodeling antibodies having an average drug binding number of about 2. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 15 shows the antitumor effect of intravenous administration of anti-EphA2 antibody and anti-EphA2 antibody-CDN conjugate (1). The black square line in the figure represents the vehicle group, the open circle line represents the anti-EphA2 antibody administration group, and the open triangle represents the anti-EphA2 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
Fig. 16 shows the antitumor effect of intravenous administration of anti-CD33 antibody and anti-CD33 antibody-CDN conjugate (1). In the figure, the black square line indicates the vehicle group, the open circle line indicates the anti-CD33 antibody administration group, and the open triangle line indicates the anti-CD33 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.
17 shows the amino acid sequence of the light chain (SEQ ID NO: 28) and the amino acid sequence of the heavy chain (SEQ ID NO: 29) of Pertuzumab.
18 shows the amino acid sequence of the light chain (SEQ ID NO: 28) and the amino acid sequence of the heavy chain (SEQ ID NO: 30) of modified anti-HER2 antibody 2.
19 shows the amino acid sequence of the light chain (SEQ ID NO: 31) and the amino acid sequence of the heavy chain (SEQ ID NO: 32) of the anti-CD33 antibody.
20 shows the amino acid sequence of the light chain (SEQ ID NO: 33) and the amino acid sequence of the heavy chain (SEQ ID NO: 34) of the anti-EphA2 antibody.
21 shows the amino acid sequence of the light chain (SEQ ID NO: 35) and the amino acid sequence of the heavy chain (SEQ ID NO: 36) of the anti-CDH6 antibody.
Fig. 22 shows the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (11) and (12). In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody 2-CDN conjugate (11) administration group, and the open circle line indicates the anti-HER2 antibody 2-CDN conjugate (12) administration group.

본 발명은, STING 아고니스트 활성을 갖는 신규 CDN 유도체 및 그 항체 약물 콘쥬게이트, 그리고 이들의 사용에 관한 것이다. 본 발명의 신규 CDN 유도체는, STING 아고니스트 활성을 갖고, 면역 세포를 부활화하여 인터페론이나 사이토카인의 산생을 유도한다. 또, 본 발명의 신규 CDN 유도체는, 당해 면역 세포의 부활화에 의해 항종양 효과를 발휘한다. 신규 CDN 유도체는, 면역 기능을 부활화하고자 하는 목적의 조직에 직접 투여되어도 되고, 또는, 표적 세포 (예를 들어, 종양 세포 또는 면역 세포) 를 인식하여 결합할 수 있는 항체에 임의의 링커를 개재하여 연결되어 전신 투여되어도 된다.The present invention relates to novel CDN derivatives having STING agonist activity, antibody drug conjugates thereof, and uses thereof. The novel CDN derivative of the present invention has STING agonist activity, activates immune cells, and induces interferon and cytokine production. In addition, the novel CDN derivative of the present invention exerts an antitumor effect by activating the immune cells. The novel CDN derivative may be directly administered to a tissue for the purpose of revitalizing immune function, or an antibody capable of recognizing and binding to a target cell (eg, tumor cell or immune cell) is interposed with an arbitrary linker. may be connected and administered systemically.

STING (Stimulator of Interferon Genes) 란, 소포체에 국재하는 막 관통형의 어댑터 단백질이다. STING 에는, 선천적인 다형 (多型) 이 높은 빈도로 존재하는 것이 알려져 있다 (PLoS One, 2013 Oct, 21, 8 (10), e77846). STING 변이형으로는, 예를 들어, 232 번째의 아미노산이 아르기닌 (R) 으로부터 히스티딘 (H) 으로 변이한 R232H 변이나, 71 번째의 아르기닌 (R) 이 히스티딘 (H) 으로, 230 번째의 글리신 (G) 이 알라닌 (A) 으로, 293 번째의 아르기닌 (R) 이 글루타민 (Q) 으로 변이한 HAQ 변이가 알려져 있다. 이와 같은 STING 의 다형에서는, STING 아고니스트 자극에 의해 유도되는 사이토카인 산생량 등의 응답의 강도에 차이가 있는 것이 알려져 있다 (Genes and I㎜unity, 2011, 12, 263-269). 그 때문에, 인간에게 있어서 안정적으로 STING 아고니스트가 작용하기 위해서는, 각 STING 의 형 (型) 에 대하여 활성을 갖는 것이 바람직하다.STING (Stimulator of Interferon Genes) is a transmembrane adapter protein localized in the endoplasmic reticulum. It is known that innate polymorphisms are present at high frequency in STING (PLoS One, 2013 Oct, 21, 8 (10), e77846). Examples of STING variants include the R232H mutation in which the 232nd amino acid is changed from arginine (R) to histidine (H), the 71st arginine (R) to histidine (H) and the 230th glycine ( A HAQ mutation in which G) is mutated to alanine (A) and the 293rd arginine (R) to glutamine (Q) is known. It is known that such polymorphisms of STING differ in the intensity of responses such as the amount of cytokine production induced by STING agonist stimulation (Genes and Immunity, 2011, 12, 263-269). Therefore, in order for the STING agonist to act stably in humans, it is preferable to have activity against each type of STING.

본 명세서에 있어서, 「암」, 「암」 및 「종양」 은 동일한 의미로 사용하고 있다.In this specification, "cancer", "cancer", and "tumor" are used with the same meaning.

본 발명에 있어서, 「면역 부활화 활성」 이란, 어떠한 형태로, 단구, 매크로파지, 수상 세포, T 세포, B 세포, NK 세포, 호중구 등의 항종양 면역에 관여하는 면역 세포의 활성화를 일으키는 것을 말하며, 예를 들어 사이토카인 및 케모카인의 산생, 면역 활성화 마커의 발현 상승, 면역 억제성 마커의 발현 저하, 세포내 시그널 전달계의 인산화 등의 변화, 유전자 발현의 변화 등, 모든 면역 세포의 구조나 기능 상의 변화를 일으키는 것을 말한다. 또, 종양 세포가 항종양 면역을 유도하는 변화를 일으키는 것도 포함하며, 예를 들어 면역 세포를 부활화 또는 유주를 유도하는 사이토카인 및 케모카인의 산생, 면역 세포에 대한 감수성 항진 등을 유도하는 것을 말한다.In the present invention, "immune activation activity" refers to activation of immune cells involved in antitumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, and neutrophils, in any form. , For example, production of cytokines and chemokines, increased expression of immune activation markers, decreased expression of immunosuppressive markers, changes in phosphorylation of intracellular signal transduction systems, changes in gene expression, etc., in all structures and functions of immune cells. It means to bring about change. It also includes changes in tumor cells that induce antitumor immunity, for example, production of cytokines and chemokines that induce activation or migration of immune cells, and induction of increased sensitivity to immune cells. .

본 발명에 있어서, 「항종양 효과」 란, 약물에 의해 종양 세포에 대하여 직접적 또는 간접적으로 영향을 미침으로써, 종양의 감소 또는 축퇴를 유도하는 것을 말한다. 예를 들어, 종양 세포에 대하여 약물이 직접적인 상해를 주거나, 종양 세포가 약물의 자극에 의해 항종양 면역을 부활화하거나, 종양 세포에 송달된 약물이 세포외로 방출되는 등 하여 종양 세포 주변의 항종양 면역이 부활화하는 것 등에 의해, 종양 세포수의 감소 및 상해, 또는 종양의 축퇴를 일으키는 것을 항종양 효과라고 한다.In the present invention, the "antitumor effect" refers to inducing reduction or regression of tumors by directly or indirectly affecting tumor cells with a drug. For example, a drug directly harms a tumor cell, a tumor cell activates anti-tumor immunity by drug stimulation, or a drug delivered to a tumor cell is released extracellularly, thereby causing anti-tumor activity around the tumor cell. Reduction of the number of tumor cells, injury, or regression of tumors due to activation of immunity or the like is called an antitumor effect.

본 발명에 있어서, 「세포 상해 활성」 이란, 어떠한 형태로, 세포에 병리적인 변화를 일으키는 것을 말하며, 직접적인 외상에 그치지 않고, DNA 의 절단이나 염기의 2량체의 형성, 염색체의 절단, 세포 분열 장치의 손상, 각종 효소 활성의 저하 등 모든 세포의 구조나 기능 상의 손상을 일으키는 것을 말한다.In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in any form, and is not limited to direct trauma, DNA cleavage, base dimer formation, chromosome cleavage, cell division device It refers to damage in the structure or function of all cells, such as damage to cells and decrease in activity of various enzymes.

본 발명에 있어서, 「세포」 에는, 동물 개체 내의 세포, 배양 세포도 포함하고 있다.In the present invention, "cells" also include cells in individual animals and cultured cells.

본 명세서에 있어서, 「할로겐 원자」 란, 불소 원자, 염소 원자, 브롬 원자, 요오드 원자를 의미한다.In this specification, a "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

본 명세서에 있어서, 「C1-C6 알킬기」 란, 탄소수 1 ∼ 6 의 직사슬 또는 분기 사슬의 알킬기를 의미한다. 「C1-C6 알킬기」 는, 총탄소수가 6 을 넘지 않으면, 알킬기 상에 시클로프로판을 갖고 있어도 된다. 「C1-C6 알킬기」 로는, 예를 들어, 이하의 구조 : In this specification, "C1-C6 alkyl group" means a C1-C6 linear or branched alkyl group. The "C1-C6 alkyl group" may have cyclopropane on the alkyl group as long as the total number of carbon atoms does not exceed 6. As a "C1-C6 alkyl group", for example, the following structure:

[화학식 47] [Formula 47]

Figure pat00047
Figure pat00047

(파선은 치환 위치를 나타낸다.) 를 들 수 있지만, 이들에 한정되지 않는다.(The broken line indicates the position of substitution.), but is not limited thereto.

본 명세서에 있어서, 「C2-C6 알케닐기」 란, 탄소수 2 ∼ 6 의 직사슬 또는 분기 사슬의 알케닐기를 의미한다.In this specification, "C2-C6 alkenyl group" means a linear or branched alkenyl group having 2 to 6 carbon atoms.

본 명세서에 있어서, 「C2-C6 알키닐기」 란, 탄소수 2 ∼ 6 의 직사슬, 분기 사슬의 알키닐기를 의미한다.In this specification, "C2-C6 alkynyl group" means a C2-C6 linear or branched alkynyl group.

본 명세서에 있어서, 「C3-C6 시클로알킬기」 란, 탄소수 3 ∼ 6 의 포화 고리형 탄화수소기를 의미한다. 「C3-C6 시클로알킬기」 는, 총탄소수가 6 을 넘지 않으면, 복수의 알킬기로 치환되어 있어도 된다. 「C3-C6 시클로알킬기」 로는, 예를 들어, 이하의 구조 : In this specification, "C3-C6 cycloalkyl group" means a C3-C6 saturated cyclic hydrocarbon group. The "C3-C6 cycloalkyl group" may be substituted with a plurality of alkyl groups as long as the total number of carbon atoms does not exceed 6. As a "C3-C6 cycloalkyl group", for example, the following structure:

[화학식 48] [Formula 48]

Figure pat00048
Figure pat00048

(파선은 치환 위치를 나타낸다.) 를 들 수 있지만, 이들에 한정되지 않는다.(A broken line indicates a substitution position.), but is not limited thereto.

본 명세서에 있어서, 「하이드록시 C1-C6 알킬기」 란, 탄소수 1 ∼ 6 의 직사슬, 분기 사슬의 알킬기의 임의의 위치에 1 또는 2 개의 하이드록시기가 치환하고 있는 알킬기를 의미한다. 「하이드록시 C1-C6 알킬기」 는, 총탄소수가 6 을 넘지 않으면, 알킬기 상에 시클로프로판을 갖고 있어도 된다. 「하이드록시 C1-C6 알킬기」 로는, 예를 들어, 이하의 구조 : In this specification, a "hydroxy C1-C6 alkyl group" means an alkyl group in which one or two hydroxy groups are substituted at an arbitrary position in a linear or branched chain alkyl group having 1 to 6 carbon atoms. The "hydroxy C1-C6 alkyl group" may have cyclopropane on the alkyl group as long as the total number of carbon atoms does not exceed 6. As a "hydroxy C1-C6 alkyl group", for example, the following structure:

[화학식 49] [Formula 49]

Figure pat00049
Figure pat00049

(파선은 치환 위치를 나타낸다.) 를 들 수 있지만, 이들에 한정되지 않는다.(The broken line indicates the position of substitution.), but is not limited thereto.

본 발명에 있어서, 「아미노 C1-C6 알킬기」 란, 탄소수 1 ∼ 6 의 직사슬 또는 분기 사슬의 알킬기의 임의의 위치에 1 또는 2 개의 아미노기가 치환하고 있는 알킬기를 의미한다. 「아미노 C1-C6 알킬기」 란, 총탄소수가 6 을 넘지 않으면, 알킬기 상에 시클로프로판을 갖고 있어도 된다. 「아미노 C1-C6 알킬기」 로는, 예를 들어, 이하의 구조 : In the present invention, "amino C1-C6 alkyl group" means an alkyl group in which one or two amino groups are substituted at an arbitrary position in a linear or branched alkyl group having 1 to 6 carbon atoms. With "amino C1-C6 alkyl group", as long as the total number of carbon atoms does not exceed 6, you may have cyclopropane on the alkyl group. Examples of the "amino C1-C6 alkyl group" include the following structures:

[화학식 50] [Formula 50]

Figure pat00050
Figure pat00050

(파선은 치환 위치를 나타낸다.) 를 들 수 있지만, 이들에 한정되지 않는다.(The broken line indicates the position of substitution.), but is not limited thereto.

＜1. 신규 CDN 유도체＞ <1. Novel CDN Derivatives>

본 발명의 신규 CDN 유도체는, 다음 식 (Ia) : The novel CDN derivative of the present invention has the following formula (Ia):

[화학식 51] [Formula 51]

Figure pat00051
Figure pat00051

로 나타내는 구조를 갖는다.has a structure represented by

L1 은, 하이드록시기, -NH2, 2-하이드록시아세틸아미노메틸기 및 2-[(2-하이드록시아세틸)아미노]에틸기로 이루어지는 군에서 선택되는 1 ∼ 3 개의 기에 의해 임의의 위치에서 치환되어 있어도 되는, 다음 식L 1 is substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxyl group, -NH 2 , a 2-hydroxyacetylaminomethyl group and a 2-[(2-hydroxyacetyl)amino]ethyl group; The following expression, which may be

[화학식 52] [Formula 52]

Figure pat00052
Figure pat00052

(여기서, (here,

R6 및 R6' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기, -NH2, C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기를 나타내고, R 6 and R 6' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 , a C1-C6 alkyl group, a C2-C6 alkenyl group or a C2-C6 alkynyl group;

R7 및 R7' 는, 각각 독립적으로, 수소 원자 또는 C1-C6 알킬기를 나타내고, 그 C1-C6 알킬기는, 할로겐 원자 및 옥소기로 이루어지는 군에서 선택되는 1 또는 2 개의 치환기에 의해 치환되어 있어도 되고, R 7 and R 7' each independently represent a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group may be substituted with 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group; ,

R8 및 R8' 는, 각각 독립적으로, 수소 원자 또는 할로겐 원자를 나타내고, R 8 and R 8' each independently represent a hydrogen atom or a halogen atom;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타내고, Z 4 represents -CH 2 -, -NH- or an oxygen atom;

Z5 는, 질소 원자 또는 -CH= 를 나타낸다) Z 5 represents a nitrogen atom or -CH=)

으로 이루어지는 군에서 선택되는 기를 나타낸다.represents a group selected from the group consisting of

또, L1 은, 하이드록시기, -NH2, 2-하이드록시아세틸아미노메틸기 및 2-[(2-하이드록시아세틸)아미노]에틸기로 이루어지는 군에서 선택되는 1 ∼ 3 개의 기에 의해 임의의 위치에서 치환되어 있어도 되는, 다음 식In addition, L 1 is an arbitrary position by 1 to 3 groups selected from the group consisting of a hydroxyl group, -NH 2 , a 2-hydroxyacetylaminomethyl group and a 2-[(2-hydroxyacetyl)amino]ethyl group. The following expression, which may be substituted in

[화학식 53] [Formula 53]

Figure pat00053
Figure pat00053

(여기서, (here,

R6 및 R6' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기, -NH2, C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기를 나타내고, R 6 and R 6' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 , a C1-C6 alkyl group, a C2-C6 alkenyl group or a C2-C6 alkynyl group;

R7 및 R7' 는, 각각 독립적으로, 수소 원자 또는 C1-C6 알킬기를 나타내고, 그 C1-C6 알킬기는, 할로겐 원자 및 옥소기로 이루어지는 군에서 선택되는 1 또는 2 개의 치환기에 의해 치환되어 있어도 되고, R 7 and R 7' each independently represent a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group may be substituted with 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group; ,

R8 및 R8' 는, 각각 독립적으로, 수소 원자 또는 할로겐 원자를 나타내고, R 8 and R 8' each independently represent a hydrogen atom or a halogen atom;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타내고, Z 4 represents -CH 2 -, -NH- or an oxygen atom;

Z5 는, 질소 원자 또는 -CH= 를 나타낸다) Z 5 represents a nitrogen atom or -CH=)

으로 이루어지는 군에서 선택되는 기를 나타낸다.represents a group selected from the group consisting of

L1 은, 바람직하게는, 다음 식으로 이루어지는 군에서 선택되는 기L 1 is preferably a group selected from the group consisting of the following formula

[화학식 54] [Formula 54]

Figure pat00054
Figure pat00054

(여기서, R9 및 R9' 는, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 9 and R 9' represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ,

R10 은, 하이드록시기, -NH2, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기를 나타내고, R 10 is a hydroxy group, -NH 2 , -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH, -CH 2 CH 2 NHC(=O)CH 2 OH, hydroxy represents a C1-C3 alkyl group or an amino C1-C3 alkyl group;

R11 및 R11' 는, 각각 독립적으로, 수소 원자, 불소 원자 혹은 메틸기를 나타내거나, 또는, R11 및 R11' 가 결합하여 시클로프로판을 나타내고, R 11 and R 11' each independently represent a hydrogen atom, a fluorine atom or a methyl group, or R 11 and R 11' combine to represent cyclopropane;

Z4 는, -CH2-, -NH- 또는 산소 원자를 나타낸다) Z 4 represents -CH 2 -, -NH- or an oxygen atom)

이다.am.

또, L1 은, 바람직하게는, 다음 식으로 이루어지는 군에서 선택되는 기In addition, L 1 is preferably a group selected from the group consisting of the following formula

[화학식 55] [Formula 55]

Figure pat00055
Figure pat00055

(여기서, R13 및 R13' 는, 각각 독립적으로, 수소 원자, 하이드록시기 또는 -NH2 를 나타내고, (Where, R 13 and R 13' each independently represent a hydrogen atom, a hydroxyl group, or -NH 2 ,

R12 는, 하이드록시기, -NH2, -CH2OH, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH 또는 -CH2CH2NHC(=O)CH2OH 를 나타내고, R 12 is a hydroxy group, -NH 2 , -CH 2 OH, -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH or -CH 2 CH 2 NHC(=O)CH 2 OH,

Z4 는, 앞에 정의한 바와 같다) Z 4 is as defined above)

이다.am.

나아가 또한, L1 은, 바람직하게는, 다음 식으로 이루어지는 군에서 선택되는 기Furthermore, L 1 is preferably a group selected from the group consisting of the following formula:

[화학식 56] [Formula 56]

Figure pat00056
Figure pat00056

(여기서, R14 는, 수소 원자 또는 -NH2 를 나타내고, (Here, R 14 represents a hydrogen atom or -NH 2 ,

R15 는, 수소 원자 또는 -C(=O)CH2OH 를 나타내고, R 15 represents a hydrogen atom or -C(=O)CH 2 OH;

R16 은, 하이드록시기, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 또는 -CH2CH2NH2 를 나타낸다) R 16 represents a hydroxy group, -NH 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 NH 2 or -CH 2 CH 2 NH 2 )

이다.am.

L1 은, 보다 바람직하게는, 다음 식으로 이루어지는 군에서 선택되는 기L 1 is more preferably a group selected from the group consisting of the following formula

[화학식 57] [Formula 57]

Figure pat00057
Figure pat00057

이다.am.

L3 은, 수소 원자, 할로겐 원자, -NH2, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기에서 선택된다.L 3 is selected from a hydrogen atom, a halogen atom, -NH 2 , a hydroxy C1-C3 alkyl group, or an amino C1-C3 alkyl group.

Q1 및 Q1' 는, 각각 독립적으로, 하이드록시기, 티올기 또는 보라노기 (BH3 -) 를 나타낸다. Q1 은, 바람직하게는, 하이드록시기 또는 티올기이다. Q1' 는, 바람직하게는, 하이드록시기 또는 티올기이다. 보다 바람직하게는, Q1 및 Q1' 의 조합은, Q1 및 Q1' 가 티올기이거나, 또는, Q1 및 Q1' 가 하이드록시기이다.Q 1 and Q 1' each independently represent a hydroxyl group, a thiol group or a borano group (BH 3 - ). Q 1 is preferably a hydroxy group or a thiol group. Q 1' is preferably a hydroxy group or a thiol group. More preferably, in the combination of Q 1 and Q 1' , Q 1 and Q 1' are a thiol group, or Q 1 and Q 1' are a hydroxy group.

Q2 및 Q2' 는, 각각 독립적으로, 산소 원자 또는 황 원자를 나타낸다. 바람직하게는, Q2 및 Q2' 는, 함께 산소 원자이거나 또는 함께 황 원자이다.Q 2 and Q 2' each independently represent an oxygen atom or a sulfur atom. Preferably, Q 2 and Q 2' are an oxygen atom together or a sulfur atom together.

Q1 및 Q2 의 조합은, 바람직하게는, Q1 이 티올기이고 또한 Q2 가 산소 원자이거나, 혹은, Q1 이 티올기이고 또한 Q2 가 황 원자이다.The combination of Q 1 and Q 2 is preferably that Q 1 is a thiol group and Q 2 is an oxygen atom, or Q 1 is a thiol group and Q 2 is a sulfur atom.

Q1' 및 Q2' 의 조합은, 바람직하게는, Q1' 가 티올기이고 또한 Q2' 가 산소 원자이거나, Q1' 가 하이드록시기이고 또한 Q2' 가 산소 원자이거나, 혹은 Q1' 가 티올기이고 또한 Q2' 가 황 원자이다.The combination of Q 1' and Q 2' is preferably such that Q 1' is a thiol group and Q 2' is an oxygen atom, Q 1' is a hydroxy group and Q 2' is an oxygen atom, or Q 1' is a thiol group and Q 2' is a sulfur atom.

X1 및 X2 는, 각각 독립적으로, 산소 원자, 황 원자 또는 -CH2- 를 나타낸다. X1 은, 바람직하게는, 산소 원자이다. X2 는, 바람직하게는, 산소 원자이다. 보다 바람직하게는, X1 및 X2 가 산소 원자이다.X 1 and X 2 each independently represent an oxygen atom, a sulfur atom or -CH 2 -. X 1 is preferably an oxygen atom. X 2 is preferably an oxygen atom. More preferably, X 1 and X 2 are oxygen atoms.

Y1 및 Y2 는, 산소 원자 또는 -CH2- 를 나타낸다. Y1 은, 바람직하게는, 산소 원자이다. Y2 는, 바람직하게는, 산소 원자이다. 보다 바람직하게는, Y1 및 Y2 가 산소 원자이다.Y 1 and Y 2 represent an oxygen atom or -CH 2 -. Y 1 is preferably an oxygen atom. Y 2 is preferably an oxygen atom. More preferably, Y 1 and Y 2 are oxygen atoms.

X3 및 X4 는, 하기 (iii) 또는 (iv) : X 3 and X 4 are the following (iii) or (iv):

(iii) Y1 이 산소 원자일 때, X3-X4 는, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (iii) when Y 1 is an oxygen atom, X 3 -X 4 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(iv) Y1 이 -CH2- 일 때, X3-X4 는, -O-CH2- 를 나타낸다, (iv) When Y 1 is -CH 2 -, X 3 -X 4 represents -O-CH 2 -;

에서 선택되는 기를 나타낸다. X3 및 X4 는, 바람직하게는, 상기 (iii) 의 -CH2-O- 이다.represents a group selected from X 3 and X 4 are preferably -CH 2 -O- of (iii) above.

X5 및 X6 은, 하기 (v) 또는 (vi) : X 5 and X 6 are (v) or (vi):

(v) Y2 가 산소 원자일 때, X5-X6 은, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는 (v) when Y 2 is an oxygen atom, X 5 -X 6 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or

(vi) Y2 가 -CH2- 일 때, X5-X6 은, -O-CH2- 를 나타낸다, (vi) When Y 2 is -CH 2 -, X 5 -X 6 represents -O-CH 2 -;

에서 선택되는 기를 나타낸다. X5 및 X6 은, 바람직하게는, 상기 (v) 의 -CH2-O- 이다.represents a group selected from X 5 and X 6 are preferably -CH 2 -O- in (v) above.

R1, R2 및 R3 은, 각각 독립적으로, 수소 원자, 할로겐 원자, -OR', -OC(=O)R', -N3, -NHR', -NR'R'' 또는 -NHC(=O)R' (여기서, R' 는, 수소 원자, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타내고, 그 C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기는, 1 내지 6 개의 할로겐 원자로 치환되어도 된다, R'' 는, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타낸다) 를 나타낸다.R 1 , R 2 and R 3 are each independently a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N 3 , -NHR', -NR'R'' or -NHC (=O)R' (wherein R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, the C1-C6 alkyl group, C2-C6 The alkenyl group or C2-C6 alkynyl group may be substituted with 1 to 6 halogen atoms, R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group) indicates

R1 은, 바람직하게는, 수소 원자, 하이드록시기 또는 불소 원자이다.R 1 is preferably a hydrogen atom, a hydroxyl group or a fluorine atom.

R2 는, 바람직하게는, 수소 원자, 하이드록시기 또는 불소 원자이다.R 2 is preferably a hydrogen atom, a hydroxyl group or a fluorine atom.

R3 은, 바람직하게는, 수소 원자, 하이드록시기 또는 불소 원자이다.R 3 is preferably a hydrogen atom, a hydroxy group or a fluorine atom.

W1 은, 질소 원자, 산소 원자, 황 원자 또는 -CH- 를 나타낸다.W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom or -CH-.

R5 는, 하기 (vii) 내지 (x) : R 5 is the following (vii) to (x):

(vii) W1 이 질소 원자일 때, R5 는 수소 원자, C1-C6 알킬기, 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타낸다 ; (vii) When W 1 is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group;

(viii) W1 이 산소 원자일 때, R5 는 존재하지 않고 ; (viii) when W 1 is an oxygen atom, R 5 is absent;

(ix) W1 이 황 원자일 때, R5 는 존재하지 않고 ; 또는 (ix) when W 1 is a sulfur atom, R 5 is absent; or

(x) W1 이 -CH- 일 때, R5 는 수소 원자, 할로겐 원자, 하이드록시기, -NH2 또는 C1-C6 알킬기를 나타낸다, (x) When W 1 is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 or a C1-C6 alkyl group;

에서 선택되는 기를 나타낸다. W1 이 질소 원자일 때, R5 는, 바람직하게는, 수소 원자이다. W1 이 -CH- 일 때, R5 는, 바람직하게는, 수소 원자이다.represents a group selected from When W 1 is a nitrogen atom, R 5 is preferably a hydrogen atom. When W 1 is -CH-, R 5 is preferably a hydrogen atom.

W2 는, 질소 원자 또는 -CH= 를 나타낸다. W2 는, 바람직하게는 -CH= 이다.W 2 represents a nitrogen atom or -CH=. W 2 is preferably -CH=.

R4 는, 수소 원자, 할로겐 원자 또는 -NH2 를 나타낸다. R4 는, 바람직하게는, 수소 원자이다.R 4 represents a hydrogen atom, a halogen atom or -NH 2 . R 4 is preferably a hydrogen atom.

Z1-Z2-Z3 은 하나가 되어, -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타내고, X 는, 할로겐 원자를 나타낸다) 또는 하기 식 중 어느 것으로 나타내는 기Z 1 -Z 2 -Z 3 become one, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -R'''-, -CH=CH-CH 2 -, -CH=CX -CH 2 -, -CX=CH-CH 2 -, -CX=CX-CH 2 -, -C(=O)-CH 2 -CH 2 -, -CH 2 -CH 2 -C(=O)- , -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )- (Where R''' represents -O- or -CH 2 -CH 2 - , X represents a halogen atom) or a group represented by any of the following formulas

[화학식 58] [Formula 58]

Figure pat00058
Figure pat00058

(여기서, 아스테리스크는, W1 에 결합하고 있는 것을 나타내고, 파선은, =C- 의 탄소 원자에 결합하고 있는 것을 나타낸다) 에서 선택되는 기를 나타낸다. Z1, Z2 및 Z3 은, 바람직하게는, 하나가 되어, -CH2-CH2-CH2-, -CH=CH-CH2-, -CH2-CH(CH3)-CH2-, -CH2-CH2-CH(CH3)-, 또는, -CH2-CH2-R'''- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타낸다) 이다.(Here, the asterisk indicates bonding to W 1 , and the broken line indicates bonding to a =C- carbon atom). Z 1 , Z 2 and Z 3 are preferably taken together, -CH 2 -CH 2 -CH 2 -, -CH=CH-CH 2 -, -CH 2 -CH(CH 3 )-CH 2 -, -CH 2 -CH 2 -CH(CH 3 )-, or -CH 2 -CH 2 -R'''- (where R''' is -O- or -CH 2 -CH 2 - represents) is.

또, 본 발명의 신규 CDN 유도체는, 바람직하게는, 다음 식 : Also, the novel CDN derivative of the present invention preferably has the following formula:

[화학식 59] [Formula 59]

Figure pat00059
Figure pat00059

으로 나타내는 구조를 갖는다.has a structure represented by

L1 은, 앞에 정의한 바와 같다.L 1 is as defined above.

Q3 및 Q3' 는, 각각 독립적으로, 하이드록시기 또는 티올기를 나타낸다. 바람직하게는, Q3 및 Q3' 는, 함께 티올기이다.Q 3 and Q 3' each independently represent a hydroxy group or a thiol group. Preferably, both Q 3 and Q 3' are thiol groups.

R21 및 R22 는, 각각 독립적으로, 하이드록시기 또는 불소 원자를 나타낸다. R21 은, 바람직하게는, 하이드록시기이다. R22 는, 바람직하게는, 불소 원자이다.R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom. R 21 is preferably a hydroxyl group. R 22 is preferably a fluorine atom.

W5 는, -NH- 또는 황 원자를 나타낸다.W 5 represents -NH- or a sulfur atom.

본 발명의 신규 CDN 유도체의 제조 방법은, 후술하는 ＜3. 제조 방법＞ 에 기재한다.The production method of the novel CDN derivative of the present invention is described in <3. Manufacturing method> described in.

＜2. 항체 약물 콘쥬게이트＞ <2. Antibody Drug Conjugates>

본 발명의 신규 CDN 유도체는, 목적으로 하는 조직에 직접 투여 (예를 들어, 종양내 투여) 되어도 되고, 혹은, 표적 세포 (예를 들어, 종양 세포 또는 면역 세포) 를 인식하여 결합할 수 있는 항체에 임의의 링커를 개재하여 연결된 항체 약물 콘쥬게이트로서 투여되어도 된다.The novel CDN derivative of the present invention may be directly administered (eg, intratumoral administration) to a target tissue, or an antibody capable of recognizing and binding to a target cell (eg, tumor cell or immune cell) may be administered as an antibody drug conjugate linked via an arbitrary linker.

본 발명의 항체 약물 콘쥬게이트는, 다음 식 (II) : The antibody drug conjugate of the present invention has the following formula (II):

[화학식 60] [Formula 60]

Figure pat00060
Figure pat00060

로 나타낸다. m1 은 항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 약물 결합수를 나타내고, Ab 는, 항체 또는 그 항체의 기능성 단편을 나타내고, L 은, Ab 와 D 를 연결하는 링커를 나타내고, D 는 상기 서술한 신규 CDN 유도체 (본 명세서 중, 신규 CDN 유도체가 항체 약물 콘쥬게이트의 일부로서 사용되는 경우, 간단히 「약물」 이라고도 칭한다) 를 나타낸다.represented by m 1 represents the number of drug bonds per antibody molecule in the antibody drug conjugate, Ab represents an antibody or a functional fragment of the antibody, L represents a linker linking Ab and D, and D represents the above description One novel CDN derivative (in this specification, when the novel CDN derivative is used as a part of an antibody drug conjugate, it is simply referred to as a "drug").

약물 D 는, 면역 세포를 부활화하는 활성, 구체적으로는, STING 아고니스트 활성을 갖는 화합물이다. 약물 D 는, 링커의 일부 또는 전부가 표적 세포 (예를 들어, 종양 세포 또는 면역 세포) 내에서 절단되면, 본래의 구조로 유리되고, 면역 부활화 효과가 발휘된다. 면역 세포에 대한 표적 세포의 감수성 항진 또는 표적 세포를 개재한 면역 세포의 부활화에 의해, 목적으로 하는 기능이 발휘된다. 목적으로 하는 기능으로는, STING 아고니스트 활성에 관련하는 기능이면 특별히 한정되지 않지만, 바람직하게는, 항종양 활성이다. 즉, 종양을 표적으로 하는 항체 (예를 들어, 항HER2 항체) 에 임의의 링커를 개재하여 연결된 약물 D 는, 표적의 세포 또는 조직에 송달되고, 링커의 일부 또는 전부가 절단되어, 면역 세포에 대한 표적 세포의 감수성 항진 또는 표적 세포를 개재한 면역 세포의 부활화 (예를 들어, 인터페론이나 사이토카인의 산생) 를 통해서 항종양 효과를 발휘한다.Drug D is a compound having an immune cell activating activity, specifically, a STING agonist activity. When part or all of the linker is cleaved in a target cell (for example, a tumor cell or an immune cell), drug D is liberated in its original structure and exhibits an immunostimulating effect. A desired function is exerted by enhancing the sensitivity of target cells to immune cells or by activating immune cells via target cells. The target function is not particularly limited as long as it is a function related to STING agonist activity, but antitumor activity is preferred. That is, drug D linked to a tumor-targeting antibody (eg, anti-HER2 antibody) via an arbitrary linker is delivered to the target cell or tissue, part or all of the linker is cleaved, and activated by immune cells. Antitumor effects are exerted through the enhancement of sensitivity of target cells to cancer or activation of immune cells via target cells (for example, production of interferons and cytokines).

본 발명의 항체 약물 콘쥬게이트에 결합되는 약물 D 는, 다음 식 (I) : Drug D coupled to the antibody drug conjugate of the present invention is the following formula (I):

[화학식 61] [Formula 61]

Figure pat00061
Figure pat00061

(여기서, (here,

L 은, L1 또는 L2 에 포함되는 임의의 -NH2 또는 하이드록시기와 결합하고, L is bonded to any -NH 2 or hydroxyl group included in L 1 or L 2 ;

L1 은, 상기 ＜1. 신규 CDN 유도체＞ 에서 규정한 바와 같고, L 1 is the above <1. New CDN derivative> as defined in

L2 는, 하기 (i) 또는 (ii) : L 2 is the following (i) or (ii):

(i) L 과 결합할 때, L2 는, -NHR', 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타내고, 여기서, R' 는, 수소 원자, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타내고, 그 C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기는, 1 내지 6 개의 할로겐 원자로 치환되어도 된다 ; 또는 (i) When combined with L, L 2 represents -NHR', a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group, wherein R' is a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alky represents a nyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group may be substituted with 1 to 6 halogen atoms; or

(ii) L 과 결합하지 않을 때, L2 는, 수소 원자 또는 할로겐 원자를 나타낸다, (ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom;

에서 선택되는 기를 나타내고, represents a group selected from

Q1, Q1', Q2, Q2', X1, X2, X3, X4, X5, X6, Y1, Y2, R1, R2, R3, R4, R5, W1, W2, Z1, Z2 및 Z3 은, 상기 ＜1. 신규 CDN 유도체＞ 에서 규정한 바와 같다.Q 1 , Q 1' , Q 2 , Q 2' , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , Y 1 , Y 2 , R 1 , R 2 , R 3 , R 4 , R 5 , W 1 , W 2 , Z 1 , Z 2 and Z 3 are the above <1. Novel CDN Derivatives> As defined in

L2 는, L 과 결합하는 경우, 바람직하게는, -NH2, -CH2NH2 또는 -CH2OH 이다. L2 는, L 과 결합하지 않는 경우, 바람직하게는, 수소 원자 또는 불소 원자이다.L 2 , when bonded to L, is preferably -NH 2 , -CH 2 NH 2 or -CH 2 OH. When L 2 is not bonded to L, it is preferably a hydrogen atom or a fluorine atom.

본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 2 식 : Drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following two formulas:

[화학식 62] [Formula 62]

Figure pat00062
Figure pat00062

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R17, R17', R18 및 R18' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고, R 17 , R 17' , R 18 and R 18' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ;

W3 은, -NH-, 산소 원자, 황 원자 또는 -CH2- 를 나타내고, W 3 represents -NH-, an oxygen atom, a sulfur atom or -CH 2 -;

W4 는, -CH= 또는 질소 원자를 나타낸다) 중 어느 것으로 나타낸다.W 4 represents -CH= or a nitrogen atom).

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 2 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following two formulas:

[화학식 63] [Formula 63]

Figure pat00063
Figure pat00063

(여기서, L1, Q1, Q1', Q2, Q2', R17, R17', R18 및 R18' 는, 앞에 정의한 바와 같다) (Where, L 1 , Q 1 , Q 1' , Q 2 , Q 2' , R 17 , R 17' , R 18 and R 18' are as defined above)

중 어느 것으로 나타낸다.indicated by which one.

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 8 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following formula 8:

[화학식 64] [Formula 64]

Figure pat00064
Figure pat00064

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,

R19, R19', R20 및 R20' 는, 각각 독립적으로, 수소 원자 또는 불소 원자를 나타낸다) R 19 , R 19' , R 20 and R 20' each independently represent a hydrogen atom or a fluorine atom)

중 어느 것으로 나타낸다. indicated by which one.

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 4 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following 4 formula:

[화학식 65] [Formula 65]

Figure pat00065
Figure pat00065

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타낸다.indicated by which one.

나아가 또한, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 4 식 : Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following 4 formula:

[화학식 66] [Formula 66]

Figure pat00066
Figure pat00066

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타낸다.indicated by which one.

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 4 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following 4 formula:

[화학식 67] [Formula 67]

Figure pat00067
Figure pat00067

(여기서, L1 은, 앞에 정의한 바와 같다) (Here, L 1 is as defined above)

중 어느 것으로 나타낸다.indicated by which one.

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 다음 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably the following formula:

[화학식 68] [Formula 68]

Figure pat00068
Figure pat00068

(여기서, L1, Q3, Q3', R21, R22 및 W5 는, 상기 ＜1. 신규 CDN 유도체＞ 에서 규정한 바와 같다) 으로 나타낸다. (Here, L 1 , Q 3 , Q 3′ , R 21 , R 22 and W 5 are as defined in <1. Novel CDN Derivative> above).

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 에 있어서, L1 은, 바람직하게는, 이하 : In addition, in the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention, L 1 is preferably:

[화학식 69] [Formula 69]

Figure pat00069
Figure pat00069

중 어느 것으로 나타낸다.indicated by which one.

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 에 있어서, L1 은, 바람직하게는, 이하의 4 식 : In addition, in the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention, L 1 is preferably of the following 4 formula:

[화학식 70] [Formula 70]

Figure pat00070
Figure pat00070

(식 중, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다) 중 어느 것으로 나타낸다.(In the formula, asterisk indicates bonding with L).

또, 본 발명의 신규 CDN 유도체 또는 본 발명의 항체 약물 콘쥬게이트에 사용되는 약물 D 는, 바람직하게는, 이하의 4 식 : In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably of the following 4 formula:

[화학식 71] [Formula 71]

Figure pat00071
Figure pat00071

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타내고, Q3, Q3' 및 W5 는, 상기 ＜1. 신규 CDN 유도체＞ 에서 규정한 바와 같다) 중 어느 것으로 나타낸다.(Here, Asterisk indicates that it is bonded to L, and Q 3 , Q 3' and W 5 are as defined in <1. Novel CDN Derivative> above).

＜2.1. 링커 구조＞ <2.1. Linker structure>

본 발명의 항체 약물 콘쥬게이트에 있어서 약물을 항체에 결합시키는 링커 구조에 대해서 설명한다. 본 발명의 항체 약물 콘쥬게이트에 사용되는 링커는, 항체와 약물을 연결시키는 링커로서 당업자가 이해하는 것이면 특별히 한정되지 않는다. 본 발명의 항체 약물 콘쥬게이트에 사용되는 링커로는, 예를 들어, Protein Cell, 2018, 9 (1) : 33-46, Pharm Res, 2015, 32 : 3526-3540, 또는, Int. J. Mol. Sci., 2016, 17, 561 에 기재되는 링커를 들 수 있지만, 이들에 한정되지 않는다. 링커는, 생체내에서 절단되는 링커여도 되고, 생체내에서 절단되지 않는 링커여도 되지만, 바람직하게는, 생체내에서 절단되는 링커이다.In the antibody drug conjugate of the present invention, a linker structure for binding a drug to an antibody will be described. The linker used in the antibody drug conjugate of the present invention is not particularly limited as long as it is understood by those skilled in the art as a linker linking the antibody and the drug. As a linker used in the antibody drug conjugate of the present invention, for example, Protein Cell, 2018, 9 (1): 33-46, Pharm Res, 2015, 32: 3526-3540, or Int. J. Mol. Although the linker described in Sci., 2016, 17, 561 is mentioned, it is not limited to these. The linker may be a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but is preferably a linker that is cleaved in vivo.

본 발명의 항체 약물 콘쥬게이트에 사용되는 링커로는, 예를 들어, 약물을 항체의 Fc 부분의 당사슬 또는 리모델링 된 당사슬에 결합시키는 (본 명세서 중, 「당사슬 콘쥬게이션」 이라고 하는 경우가 있다) 링커 (예를 들어, WO2018/003983 에 기재된다), 또는, 약물을 항체의 임의의 아미노산 잔기 (예를 들어, 시스테인 잔기 또는 리신 잔기) 에 결합시키는 링커 (예를 들어, WO2014/057687 에 기재된다) 를 들 수 있지만, 이들에 한정되지 않는다. 약물을 항체의 임의의 아미노산 잔기에 결합시키는 링커로는, 바람직하게는, Ab 의 시스테인의 술프하이드릴기 (SH 기) 와 티오에테르 결합하는 경우 (본 명세서 중, 「시스테인 콘쥬게이션」 이라고 하는 경우가 있다) 또는 Ab 의 리신의 아미노기 (NH2 기) 와 아미드 결합하는 경우 (본 명세서 중, 「리신 콘쥬게이션」 이라고 하는 경우가 있다) 를 들 수 있으며, 바람직하게는, 시스테인 콘쥬게이션이다.As the linker used in the antibody drug conjugate of the present invention, for example, a linker that binds a drug to a sugar chain or a remodeled sugar chain of the Fc portion of an antibody (in this specification, it may be referred to as "sugar chain conjugation") (eg, as described in WO2018/003983), or a linker that connects the drug to any amino acid residue (eg, a cysteine residue or a lysine residue) of an antibody (eg, as described in WO2014/057687) but is not limited thereto. The linker for binding the drug to any amino acid residue of the antibody is preferably a thioether bond with the sulfhydryl group (SH group) of the cysteine of Ab (referred to as "cysteine conjugation" in this specification) ) or an amide bond with the lysine amino group (NH 2 group) of Ab (sometimes referred to as “lysine conjugation” in the present specification), preferably cysteine conjugation.

본 발명의 바람직한 링커 L 은, 다음의 식으로 나타낸다. A preferred linker L of the present invention is represented by the following formula.

-Lb-La-Lp-Lc-* -Lb-La-Lp-Lc-*

(여기서, 아스테리스크는, 약물 D 의 L1 또는 L2 에 포함되는 임의의 아미노기 또는 하이드록시기와 결합하고 있는 것을 나타낸다.) 로 나타낸다.(Here, Asterisk indicates that it is bonded to any amino group or hydroxyl group contained in L 1 or L 2 of drug D.).

먼저, Lp 에 대해서 설명한다.First, Lp will be described.

Lp 는, 생체내 또는 표적 세포에 있어서 절단 가능한 아미노산 서열로 이루어지는 링커 (이하, 본 명세서 중, 펩티드 링커라고도 한다) 를 나타내거나, 또는 존재하지 않는다.Lp represents a linker composed of an amino acid sequence cleavable in vivo or in a target cell (hereinafter, also referred to as a peptide linker in the present specification), or does not exist.

Lp 는, 예를 들어, 펩티다아제나 에스테라아제 등의 효소의 작용에 의해, 절단된다. Lp 는, 2 내지 7 개 (바람직하게는, 2 내지 4 개) 의 아미노산으로 구성되는 펩티드이다. Lp 는, 그 N 말단에 있어서 후술하는 La 의 우단의 카르보닐기와 아미드 결합을 형성하고, C 말단에 있어서 Lc 의 아미노기 (-NH-) 와 아미드 결합을 형성한다. 상기 펩티다아제 등의 효소에 의해, Lp 의 C 말단측의 아미드 결합이 절단된다.Lp is cleaved, for example, by the action of enzymes such as peptidase and esterase. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond with the carbonyl group at the right end of La, which will be described later, at its N-terminus, and forms an amide bond with an amino group (-NH-) of Lc at its C-terminus. The amide bond at the C-terminal side of Lp is cleaved by an enzyme such as the above peptidase.

Lp 를 구성하는 아미노산은 특별히 한정되지는 않지만, 예를 들어, L- 또는 D-아미노산이며, 바람직하게는 L-아미노산이다. 또, α-아미노산 외에, β-알라닌, ε-아미노카프로산, γ-아미노부티르산 등의 구조의 아미노산이어도 되고, 나아가서는, 예를 들어, N-메틸화 된 아미노산 등의 비천연형의 아미노산이어도 된다. Lp 의 아미노산 서열은, 특별히 한정되지 않지만, 구성하는 아미노산으로서, 글리신 (Gly ; G), 발린 (Val ; V), 알라닌 (Ala ; A), 페닐알라닌 (Phe ; F), 글루타민산 (Glu ; E), 이소류신 (Ile ; I), 프롤린 (Pro ; P), 시트룰린 (Cit), 류신 (Leu ; L), 메티오닌 (Met ; M), 세린 (Ser ; S), 리신 (Lys ; K) 및 아스파르트산 (Asp ; D) 등을 들 수 있다. 이들 중에서 바람직하게는, 글리신 (Gly ; G), 발린 (Val ; V), 알라닌 (Ala ; A), 페닐알라닌 (Phe : F), 시트룰린 (Cit) 이다. 이들 아미노산은 중복해도 되며, 임의로 선택된 아미노산을 포함하는 아미노산 서열을 갖는다. 또, 아미노산의 종류에 따라, 약물 유리의 패턴을 컨트롤할 수 있다.The amino acid constituting Lp is not particularly limited, but is, for example, L- or D-amino acid, preferably L-amino acid. In addition to α-amino acids, amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may be used, and furthermore, non-natural amino acids such as N-methylated amino acids may be used. . The amino acid sequence of Lp is not particularly limited, but as constituting amino acids, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E) , isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), methionine (Met; M), serine (Ser; S), lysine (Lys; K) and aspartic acid. (Asp; D) etc. are mentioned. Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe: F), and citrulline (Cit) are preferred. These amino acids may overlap and have an amino acid sequence containing arbitrarily selected amino acids. In addition, the pattern of drug release can be controlled according to the type of amino acid.

Lp 의 구체예로는, 예를 들어, -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-, -ICit- 를 들 수 있다. 링커 Lp 는, 바람직하게는, -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, -FCit- 이다. 링커 Lp 는, 보다 바람직하게는, -GGVA-, -GGFG, -GGVCit- 이다. 또, 링커 Lp 는, 바람직하게는, -GGFG- 또는 -GGPI- 이다.Specific examples of Lp include, for example, -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK- , -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-, -ICit-. Linker Lp is preferably -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, or -FCit-. Linker Lp is more preferably -GGVA-, -GGFG, or -GGVCit-. Moreover, linker Lp is preferably -GGFG- or -GGPI-.

다음으로, La 에 대해서 설명한다.Next, La is explained.

La 는, 이하 : La is, below:

-C(=O)-(CH2CH2)n2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-,

-C(=O)-(CH2CH2)n2-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -CH 2 -C(=O)-,

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-,

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -CH 2 -C(=O)-,

-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2O)n3-CH2-C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-;

-(CH2)n4-O-C(=O)-, 및, -(CH 2 )n 4 -OC(=O)-, and

-(CH2)n9-C(=O)- -(CH 2 )n 9 -C(=O)-

(여기서, 식 중, n2 는 1 ∼ 3 의 정수 (바람직하게는, 1 또는 2) 를 나타내고, n3 은 1 ∼ 5 의 정수 (바람직하게는, 2 ∼ 5 의 정수, 보다 바람직하게는, 3 또는 4) 를 나타내고, n4 는 0 ∼ 2 의 정수 (바람직하게는, 0 또는 1) 를 나타내고, n9 는 2 ∼ 7 의 정수 (바람직하게는, 2 ∼ 5 의 정수, 보다 바람직하게는, 2, 3 또는 5) 를 나타낸다) 로 이루어지는 군에서 선택되는 어느 1 개를 나타낸다.(Where, in the formula, n 2 represents an integer of 1 to 3 (preferably 1 or 2), n 3 is an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably, 3 or 4), n 4 represents an integer of 0 to 2 (preferably 0 or 1), and n 9 represents an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably , 2, 3 or 5) is shown.

La 는, 바람직하게는, 이하 : La is preferably:

-C(=O)-CH2CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 4 -CH 2 -C(=O)-,

-C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -CH 2 -C(=O)-,

-CH2-OC(=O)-, -CH 2 -OC(=O)-,

-OC(=O)-, -OC(=O)-,

-(CH2)5-C(=O)-, 및, -(CH 2 ) 5 -C(=0)-, and

로 이루어지는 군에서 선택되는 어느 1 개를 나타낸다.It shows any one selected from the group which consists of.

La 는, 보다 바람직하게는, La is more preferably,

-C(=O)-CH2CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-,

-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-,

또는 or

-(CH2)5-C(=O)- -(CH 2 ) 5 -C(=O)-

이다.am.

La 는, 보다 더 바람직하게는, -C(=O)-CH2CH2-C(=O)- 이다.La is more preferably -C(=O)-CH 2 CH 2 -C(=O)-.

다음으로, Lb 에 대해서 설명한다.Next, Lb is explained.

Lb 는, 당사슬 콘쥬게이션의 링커에 사용되는 스페이서 (본 명세서 중, 「당사슬 콘쥬게이션의 링커의 스페이서」 라고도 한다), 또는, 시스테인 콘쥬게이션에 사용되는 스페이서 (본 명세서 중, 「시스테인 콘쥬게이션의 링커의 스페이서」 라고도 한다) 를 나타낸다.Lb is a spacer used for linker of sugar chain conjugation (in this specification, also referred to as "spacer for linker of sugar chain conjugation") or a spacer used for cysteine conjugation (in this specification, "linker for cysteine conjugation") It is also referred to as "the spacer of").

＜Lb 가 「당사슬 콘쥬게이션의 링커의 스페이서」 인 경우＞ <When Lb is "linker spacer of sugar chain conjugation">

Lb 가 「당사슬 콘쥬게이션의 링커의 스페이서」 인 경우, Lb 는, 특별히 한정되지 않지만, 예를 들어, 다음 식으로 나타내는 스페이서를 들 수 있다.When Lb is "a linker spacer of sugar chain conjugation", Lb is not particularly limited, but examples thereof include a spacer represented by the following formula.

[화학식 72][Formula 72]

Figure pat00072
Figure pat00072

[화학식 73] [Formula 73]

Figure pat00073
Figure pat00073

또는,or,

[화학식 74][Formula 74]

Figure pat00074
Figure pat00074

상기에서 나타내는 각각의 구조식에 있어서, 아스테리스크 (*) 는 La 의 좌단의 -(C=O)- 또는 -CH2- 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 당사슬 또는 리모델링 된 당사슬과 결합하고 있는 것을 나타낸다.In each of the above structural formulas, the asterisk (*) indicates bonding to -(C=O)- or -CH 2 - at the left end of La, and the broken line bonds to the Ab sugar chain or remodeled sugar chain. indicates what you are doing

Lb 에 Lb-1, Lb-2 또는 Lb-3 중 어느 것을 선택하는 경우, 트리아졸 고리 부위는, 기하 이성 구조를 갖고, 1 개의 Lb 중에, 2 종류의 구조 중 어느 일방을 포함하거나, 또는, 그들의 혼합물을 포함한다. 본 발명의 항체 약물 콘쥬게이트는, 항체 1 분자에 복수의 약물을 결합시킬 수 있다. 항체 1 분자에 복수의 약물을 결합시키는 경우, Lb 도 복수 존재하게 된다 (예를 들어, 후술하는 ＜3. 제조 방법＞ 의 E 법에 나타낸 항체 약물 콘쥬게이트의 모식도 (1e) 를 참조할 것). Lb 가 Lb-1, Lb-2 또는 Lb-3 중 어느 것에서 선택되고, 그 Lb 가 항체 1 분자에 대하여 복수 존재하는 경우 (예를 들어, 후술하는 m2 가 1 또는 2 인 경우), 각각의 Lb 에 있어서, 트리아졸 고리 부위는 기하 이성 구조를 갖고, 1 개의 Lb 중에, 2 종류의 구조 중 어느 일방을 포함하거나, 또는, 그들의 혼합물을 포함한다.When Lb is selected from Lb-1, Lb-2 or Lb-3, the triazole ring moiety has a geometric isomer structure and contains either one of the two types of structures in one Lb, or including mixtures thereof. In the antibody drug conjugate of the present invention, a plurality of drugs can be bound to one antibody molecule. When a plurality of drugs are bound to one antibody molecule, a plurality of Lb will also be present (for example, refer to the schematic diagram (1e) of the antibody drug conjugate shown in method E of <3. Manufacturing method> described later) . When Lb is selected from any of Lb-1, Lb-2, or Lb-3, and the Lb exists in plural with respect to one antibody molecule (for example, when m 2 described later is 1 or 2), each In Lb, the triazole ring moiety has a geometric isomeric structure, and contains either one of two types of structures or a mixture thereof in one Lb.

＜Lb 가 「시스테인 콘쥬게이션의 링커의 스페이서」 인 경우＞ <When Lb is "cysteine conjugated linker spacer">

Lb 가, 「시스테인 콘쥬게이션의 링커의 스페이서」 인 경우, Lb 는, 특별히 한정되지 않지만, 예를 들어, -(숙신이미드-3-일-N)- 를 들 수 있다. 본 발명에 있어서, 「-(숙신이미드-3-일-N)-」 는, 다음 식 : When Lb is "a spacer for a cysteine conjugated linker", Lb is not particularly limited, and examples thereof include -(succinimide-3-yl-N)-. In the present invention, "-(succinimide-3-yl-N)-" is the following formula:

[화학식 75] [Formula 75]

Figure pat00075
Figure pat00075

으로 나타내는 구조를 갖는다. 상기에서 나타내는 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 항체의 시스테인 잔기의 측사슬과 티오에테르를 형성하여 결합하고 있다.has a structure represented by In the structural formula shown above, the asterisk represents bonding to La, and the broken line represents bonding by forming a thioether with the side chain of the cysteine residue of the antibody.

다음으로, Lc 에 대해서 설명한다.Next, Lc is explained.

Lc 는, -NH-CH2-, -페닐기-CH2-O(C=O)- 또는 -NH-헤테로아릴기-CH2-O(C=O)- 를 나타내거나, 혹은, 존재하지 않는다. 여기서, 페닐기로는, 바람직하게는, 1,4-페닐기이고, 헤테로아릴기로서 바람직하게는, 2,5-피리딜기, 3,6-피리딜기, 2,5-피리미딜기 또는 2,5-티에닐기이다. Lc 는, 바람직하게는, -NH-CH2- 이거나, 또는 존재하지 않는다.Lc represents -NH-CH 2 -, -phenyl group-CH 2 -O(C=O)-, or -NH-heteroaryl group-CH 2 -O(C=O)-, or does not exist . Here, the phenyl group is preferably a 1,4-phenyl group, and the heteroaryl group is preferably a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group or a 2,5-pyridyl group. -It is a thienyl group. Lc is preferably -NH-CH 2 - or absent.

본 발명의 보다 바람직한 링커 L 은, A more preferred linker L of the present invention is,

약물과 항체의 결합 양식이 「당사슬 콘쥬게이션」 인 경우, When the binding mode of the drug and antibody is "sugar chain conjugation",

-ZL1-C(=O)-CH2CH2-C(=O)-GGFG-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGVA-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGVCit-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGFCit-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFCit-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGICit-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGICit-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGFM-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFM-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGPI-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGLM-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGLM-,

-ZL1-C(=O)-CH2CH2-C(=O)-FG-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-FG-,

-ZL1-C(=O)-CH2CH2-C(=O)-VA-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-VA-,

-ZL1-C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -,

-ZL1-C(=O)-CH2CH2-C(=O)-GGVA-NH-CH2 -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-CH 2

-ZL1-C(=O)-CH2CH2-C(=O)-GGVCit-NH-CH2-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-CH 2 -,

-ZL1-C(=O)-CH2CH2-C(=O)-GGFCit-NH-CH2-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFCit-NH-CH 2 -,

-ZL1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, 또는 -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-, or

-ZL1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 4 -CH 2 -C(=O)-,

(여기서, ZL1 은, 상기 Lb 의 이하로 나타내는 구조식 : (Here, Z L1 is a structural formula represented by the following of the above Lb:

[화학식 76] [Formula 76]

Figure pat00076
Figure pat00076

을 나타낸다) 이고, 혹은, represents) and, or,

약물과 항체의 결합 양식이 「시스테인 콘쥬게이션」 인 경우, When the binding mode of the drug and antibody is "cysteine conjugation",

-ZL2-(CH2)5-C(=O)-GGFG-, -Z L2 -(CH 2 ) 5 -C(=O)-GGFG-,

-ZL2-(CH2)5-C(=O)-GGVA-, -Z L2 -(CH 2 ) 5 -C(=O)-GGVA-,

-ZL2-(CH2)5-C(=O)-GGVCit-, -Z L2 -(CH 2 ) 5 -C(=O)-GGVCit-,

-ZL2-(CH2)5-C(=O)-GGFCit-, -Z L2 -(CH 2 ) 5 -C(=O)-GGFCit-,

-ZL2-(CH2)5-C(=O)-GGICit-, -Z L2 -(CH 2 ) 5 -C(=O)-GGICit-,

-ZL2-(CH2)5-C(=O)-GGFM-, -Z L2 -(CH 2 ) 5 -C(=O)-GGFM-,

-ZL2-(CH2)5-C(=O)-GGPI-, -Z L2 -(CH 2 ) 5 -C(=O)-GGPI-,

-ZL2-(CH2)5-C(=O)-GGLM-, -Z L2 -(CH 2 ) 5 -C(=O)-GGLM-,

-ZL2-(CH2)5-C(=O)-FG-, -Z L2 -(CH 2 ) 5 -C(=O)-FG-,

-ZL2-(CH2)5-C(=O)-VA-, -Z L2 -(CH 2 ) 5 -C(=O)-VA-,

-ZL2-(CH2)5-C(=O)-GGFG-NH-CH2-, -Z L2 -(CH 2 ) 5 -C(=O)-GGFG-NH-CH 2 -,

-ZL2-(CH2)5-C(=O)-GGVA-NH-CH2-, -Z L2 -(CH 2 ) 5 -C(=O)-GGVA-NH-CH 2 -,

-ZL2-(CH2)5-C(=O)-GGVCit-NH-CH2-, -Z L2 -(CH 2 ) 5 -C(=O)-GGVCit-NH-CH 2 -,

-ZL2-(CH2)5-C(=O)-GGFCit-NH-CH2-, -Z L2 -(CH 2 ) 5 -C(=O)-GGFCit-NH-CH 2 -,

-ZL2-(CH2)5-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, 또는 -Z L2 -(CH 2 ) 5 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-, or

-ZL2-(CH2)5-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -Z L2 -(CH 2 ) 5 -C(=O)-NH-(CH 2 CH 2 O) 4 -CH 2 -C(=O)-,

(여기서, ZL2 는, 상기 Lb 의 이하로 나타내는 구조식 : (Here, Z L2 is a structural formula represented by the following of the above Lb:

[화학식 77] [Formula 77]

Figure pat00077
Figure pat00077

으로 나타내는 -(숙신이미드-3-일-N)- 를 나타낸다.) Represents -(succinimide-3-yl-N)- represented by .)

이다.am.

본 발명의 보다 바람직한 링커 L 은, 약물과 항체의 결합 양식이 「당사슬 콘쥬게이션」 이며, A more preferred linker L of the present invention has a drug-antibody binding mode of "sugar chain conjugation",

-ZL1-C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -,

또는 or

-ZL1-C(=O)-CH2CH2-C(=O)-GGPI-NH-CH2-, -Z L1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-CH 2 -,

(여기서, ZL1 은, 상기 Lb 의 이하로 나타내는 구조식 : (Here, Z L1 is a structural formula represented by the following of the above Lb:

[화학식 78] [Formula 78]

Figure pat00078
Figure pat00078

을 나타낸다) 이다.represents).

상기의 「바람직한 링커 L」 및 「보다 바람직한 링커 L」 에 있어서의 우단은, 식 (I) 의 L1 또는 L2 에 포함되는 임의의 -NH2 또는 하이드록시기와 결합하고 있다.The right end in the above "preferred linker L" and "more preferred linker L" is bonded to any -NH 2 or hydroxyl group contained in L 1 or L 2 of formula (I).

＜2.2. 항체 및 그 당사슬 수식＞ <2.2. Antibody and its sugar chain modification>

＜2.2.1 항체＞ <2.2.1 Antibodies>

본 명세서에 있어서, 「유전자」 란, 단백질의 아미노산을 코드하는 뉴클레오티드 서열이 포함되는 뉴클레오티드 혹은 뉴클레오티드 서열, 또는 그 상보 사슬을 의미하며, 예를 들어, 단백질의 아미노산을 코드하는 뉴클레오티드 서열이 포함되는 뉴클레오티드 서열 또는 그 상보 사슬인 폴리 뉴클레오티드, 올리고 뉴클레오티드, DNA, mRNA, cDNA, RNA 등은 「유전자」 의 의미에 포함된다.As used herein, "gene" means a nucleotide or nucleotide sequence containing a nucleotide sequence encoding an amino acid of a protein, or a complementary chain thereof, for example, a nucleotide sequence containing a nucleotide sequence encoding an amino acid of a protein A sequence or its complementary chain, such as polynucleotide, oligonucleotide, DNA, mRNA, cDNA, RNA, etc., is included in the meaning of "gene".

본 명세서에 있어서, 「뉴클레오티드」, 「폴리 뉴클레오티드」 또는 「뉴클레오티드 서열」 과「핵산」 은 동일한 의미이며, 예를 들어, DNA, RNA, 프로브, 올리고 뉴클레오티드, 폴리 뉴클레오티드, 프라이머 등도 「뉴클레오티드」 또는 「뉴클레오티드 서열」 의 의미에 포함된다.In the present specification, "nucleotide", "polynucleotide" or "nucleotide sequence" and "nucleic acid" have the same meaning, and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also referred to as "nucleotide" or "nucleotide sequence". It is included in the meaning of "nucleotide sequence".

본 명세서에 있어서는, 「폴리펩티드」, 「펩티드」, 「단백질」 은 구별하지 않고 사용하고 있다.In this specification, "polypeptide", "peptide", and "protein" are used without distinction.

본 명세서에 있어서, 「항체의 기능성 단편」 이란, 「항체의 항원 결합 단편」 이라고도 불리고, 항원과의 결합 활성을 갖는 항체의 부분 단편을 의미하고 있으며, Fab, F(ab')2, Fv, scFv, diabody, 선상 항체 및 항체 단편으로부터 형성된 다특이성 항체 등을 포함한다. 또, F(ab')2 를 환원 조건하에서 처리한 항체의 가변 영역의 1 가의 단편인 Fab' 도 항체의 항원 결합 단편에 포함된다. 단, 항원과의 결합능을 갖고 있는 한 이들 분자에 한정되지 않는다. 또, 이들 항원 결합 단편에는, 항체 단백질의 전체 길이 분자를 적당한 효소로 처리한 것 뿐만 아니라, 유전자 공학에 의해 개변된 항체 유전자를 사용하여 적당한 숙주 세포에 있어서 산생된 단백질도 포함된다.In the present specification, "functional antibody fragment" is also called "antigen-binding fragment of antibody", and means a partial fragment of an antibody having antigen-binding activity, Fab, F(ab')2, Fv, scFvs, diabodies, linear antibodies and multispecific antibodies formed from antibody fragments, and the like. In addition, Fab', which is a monovalent fragment of the variable region of an antibody treated with F(ab')2 under reducing conditions, is also included in the antigen-binding fragment of an antibody. However, it is not limited to these molecules as long as it has antigen-binding ability. Further, these antigen-binding fragments include not only those obtained by treating the full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using antibody genes modified by genetic engineering.

본 발명의 기능성 단편은, IgG 중사슬의 Fc 영역에 있어서 잘 보존된 N 결합형 당사슬에 의한 수식을 받는 아스파라긴 (Asn297) 및 그 주변의 아미노산을 유지하고, 또한 항원과의 결합능을 갖고 있는 기능성 단편을 포함한다.The functional fragment of the present invention is a functional fragment that retains asparagine (Asn297) and its surrounding amino acids that are modified by an N-linked sugar chain, which is well conserved in the Fc region of the heavy chain of IgG, and has antigen-binding ability. includes

본 발명의 항체 약물 콘쥬게이트에 사용되는 항체는, 면역 글로블린을 의미하며, 항원과 면역 특이적으로 결합하는 항원 결합 부위를 함유하는 분자이다. 본 발명의 항체로서, IgG, IgE, IgM, IgD, IgA 및 IgY 중 어느 클래스여도 되지만, IgG 가 바람직하다. 또, 서브 클래스로서, IgG1, IgG2, IgG3, IgG4, IgA1 및 IgA2 중 어느 것이어도 되지만, IgG1, IgG2 또는 IgG4 가 바람직하다 (IgG 중사슬의 Fc 영역에 ADCC 및 ADCP 활성에 영향을 미치는 변이를 갖는 항체를 포함한다).The antibody used in the antibody drug conjugate of the present invention refers to an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. As the antibody of the present invention, any class of IgG, IgE, IgM, IgD, IgA and IgY may be used, but IgG is preferred. Further, the subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, but IgG1, IgG2 or IgG4 is preferable (having mutations affecting ADCC and ADCP activity in the Fc region of the IgG heavy chain) antibodies).

본 발명의 항체의 아이소타입으로서 IgG1 을 사용하는 경우에는, 정상 영역의 아미노산 잔기의 일부를 치환함으로써, 이펙터 기능을 조정하는 것이 가능하다 (WO88/07089, WO94/28027, WO94/29351 참조). IgG1 의 변이체로는, 예를 들어, IgG1 LALA 변이 (IgG1-L234A, L235A) 를 들 수 있다. 상기 L234A, L235A 는 EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No.1 (May 15, 1969), pp. 78-85) 에 의해 특정되는 234 위치 및 235 위치의 류신의 알라닌으로의 치환을 나타낸다.When IgG1 is used as the isotype of the antibody of the present invention, it is possible to adjust the effector function by substituting a part of amino acid residues in the constant region (see WO88/07089, WO94/28027, WO94/29351). Examples of IgG1 variants include IgG1 LALA mutations (IgG1-L234A, L235A). The L234A and L235A are positions 234 and 235 specified by the EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85). Indicates a substitution of leucine for alanine at position.

항체는, 어느 종에서 유래해도 되지만, 바람직하게는, 인간, 래트, 마우스 및 토끼를 예시할 수 있다. 인간 이외의 종에서 유래하는 경우에는, 주지의 기술을 이용하여, 키메라화 또는 인간화하는 것이 바람직하다. 본 발명의 항체는, 폴리클로날 항체여도 되고, 모노클로날 항체여도 되지만, 모노클로날 항체가 바람직하다. 모노클로날 항체에는, 래트 항체, 마우스 항체, 토끼 항체 등의 비인간 동물 유래의 모노클로날 항체, 키메라 항체, 인간화 항체, 인간 항체, 그들의 기능성 단편, 또는 그들의 수식체가 포함된다.Antibodies may be derived from any species, but are preferably exemplified by humans, rats, mice and rabbits. When derived from a species other than human, it is preferably chimerized or humanized using known techniques. The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, and modifications thereof.

항체는, 바람직하게는, 종양 세포 또는 면역 세포를 표적으로 하는 항체이지만, 이들에 한정되지 않는다. 항체는, 보다 바람직하게는, 종양 세포를 표적으로 하는 항체이다.Antibodies are preferably antibodies targeting tumor cells or immune cells, but are not limited thereto. The antibody is more preferably an antibody targeting tumor cells.

종양 세포를 표적으로 하는 항체가 사용되는 경우, 항체로는, 종양 세포를 인식할 수 있는 특성, 종양 세포에 결합할 수 있는 특성, 종양 세포내에 도입되어 내재화하는 특성, 나아가서는 종양 세포를 상해하는 특성 중 1 또는 그 이상의 특성을 구비하고 있는 것이 바람직하다. 본 발명의 항체에 링커를 개재하여 결합되는 약물은, STING 아고니스트 활성을 갖는다. 본 발명의 약물은 인터페론 제어 인자 3 (interferon regulatory factor-3 (IRF3)) 의 시그널을 활성화하여 인터페론을 유도한다. 따라서, 본 발명의 항체 약물 콘쥬게이트에 종양 세포를 표적으로 하는 항체가 사용되는 경우, 항체 약물 콘쥬게이트는 체내에 투여된 후, 종양 부위에 송달되어, 종양 세포내에 도입된 후 펩티다아제 등에 의해 링커 부분이 절단되어 약물 부분이 유리된다. 유리된 약물 부분은 STING 아고니스트 활성을 통해서 항종양 면역을 부활화하여, 항종양 효과를 발휘시키는 것으로 생각된다.When an antibody targeting tumor cells is used, the antibody has characteristics capable of recognizing tumor cells, characteristics capable of binding to tumor cells, characteristics of being introduced into and internalizing tumor cells, and furthermore, having characteristics capable of injuring tumor cells. It is preferable to have one or more of the characteristics. A drug linked to the antibody of the present invention via a linker has a STING agonist activity. The drug of the present invention induces interferon by activating the signal of interferon regulatory factor-3 (IRF3). Therefore, when an antibody targeting a tumor cell is used in the antibody drug conjugate of the present invention, the antibody drug conjugate is administered into the body, delivered to the tumor site, introduced into the tumor cell, and then the linker portion by peptidase or the like. This cleavage liberates the drug moiety. It is thought that the free drug moiety activates antitumor immunity through STING agonist activity and exerts an antitumor effect.

항체의 종양 세포에 대한 결합성은, 플로 사이토메트리를 사용하여 확인할 수 있다. 종양 세포내로의 항체의 도입은, (1) 치료 항체에 결합하는 2 차 항체 (형광 표지) 를 사용하여 세포내에 도입된 항체를 형광 현미경으로 가시화하는 어세이 (Cell Death and Differentiation (2008) 15, 751-761), (2) 치료 항체에 결합하는 2 차 항체 (형광 표지) 를 사용하여 세포내에 도입된 형광량을 측정하는 어세이 (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004) 또는 (3) 치료 항체에 결합하는 이뮤노톡신을 사용하여, 세포내에 도입되면 독소가 방출되어 세포 증식이 억제된다는 Mab-ZAP 어세이 (Bio Techniques 28 : 162-165, January 2000) 를 사용하여 확인할 수 있다. 이뮤노톡신으로는, 디프테리아 독소의 촉매 영역과 프로테인 G 의 리콤비넌트 복합 단백질도 사용 가능하다.The antibody's binding ability to tumor cells can be confirmed using flow cytometry. The introduction of antibodies into tumor cells is (1) an assay in which the antibody introduced into cells is visualized under a fluorescence microscope using a secondary antibody (fluorescent label) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15; 751-761), (2) Assay for measuring the amount of fluorescence introduced into cells using a secondary antibody (fluorescent label) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004 ) or (3) using an immunotoxin that binds to a therapeutic antibody, using the Mab-ZAP assay (Bio Techniques 28: 162-165, January 2000) that the toxin is released and cell proliferation is inhibited when introduced into cells You can check. As the immunotoxin, a recombinant complex protein of the catalytic domain of diphtheria toxin and protein G can also be used.

본 발명에 있어서, 「높은 내재화능」 이란, 당해 항체와 사포린 표지 항마우스 또는 래트 IgG 항체를 첨가한 표적 항원 발현 세포 (예를 들어, 항HER2 항체를 사용하는 경우에는, HER2 발현 세포) 의 생존률 (항체 미첨가 시의 세포 생존률을 100 ％ 로 한 상대율로 나타낸 것) 이, 바람직하게는 70 ％ 이하, 보다 바람직하게는 60 ％ 이하인 것을 의미한다.In the present invention, "high internalization ability" means target antigen-expressing cells (for example, HER2-expressing cells when anti-HER2 antibody is used) to which the antibody and saporin-labeled anti-mouse or rat IgG antibody are added. It means that the survival rate (expressed as a relative rate with the cell viability when no antibody is added as 100%) is preferably 70% or less, more preferably 60% or less.

본 발명의 항체 약물 콘쥬게이트에 종양 세포를 표적으로 한 항체를 사용하는 경우, 항체 자체가 항종양 효과를 갖는 것은, 바람직하지만, 필수는 아니다. 본 발명의 항체 약물 콘쥬게이트에 사용되는 항체는, 종양 세포내로 이행하는 내재화의 성질을 갖는 것이 바람직하다.When using an antibody targeting tumor cells in the antibody drug conjugate of the present invention, it is desirable, but not essential, that the antibody itself has an antitumor effect. The antibody used in the antibody drug conjugate of the present invention preferably has the property of internalization to migrate into tumor cells.

약물 그리고 항체 약물 콘쥬게이트의 항종양 활성은, 종양 세포에 대한 세포 상해 활성, 항세포 효과, 종양 체적의 축퇴를 말한다. 공지된 in vitro 또는 in vivo 의 평가계를 사용하여, 항종양 활성을 확인할 수 있다.Antitumor activity of drugs and antibody drug conjugates refers to cytotoxic activity against tumor cells, anticellular effect, and reduction of tumor volume. The antitumor activity can be confirmed using a known in vitro or in vivo evaluation system.

약물 그리고 항체 약물 콘쥬게이트의 면역 부활화 활성은, 면역 세포에 대한 종양 세포의 감수성 항진 또는 종양 세포를 개재한 면역 세포의 부활화를 말한다. 공지된 in vitro 또는 in vivo 의 평가계를 사용하여, 면역 부활화 활성을 확인할 수 있다.The immunopotentiation activity of drugs and antibody-drug conjugates refers to enhancement of sensitivity of tumor cells to immune cells or activation of immune cells via tumor cells. The immunostimulating activity can be confirmed using a known in vitro or in vivo evaluation system.

본 발명에 사용되는 항체로는, 예를 들어, 항HER2 항체, 항HER3 항체, 항DLL3 항체, 항FAP 항체, 항CDH11 항체, 항CDH6 항체, 항A33 항체, 항CanAg 항체, 항CD19 항체, 항CD20 항체, 항CD22 항체, 항CD30 항체, 항CD33 항체, 항CD56 항체, 항CD70 항체, 항CD98 항체, 항TROP2 항체, 항CEA 항체, 항Cripto 항체, 항EphA2 항체, 항G250 항체, 항MUC1 항체, 항GPNMB 항체, 항Integrin 항체, 항PSMA 항체, 항Tenascin-C 항체, 항SLC44A4 항체, 항Mesothelin 항체, 항ENPP3 항체, 항CD47 항체, 항EGFR 항체, 항GPR20 항체 또는 항DR5 항체를 들 수 있지만, 이들에 한정되지 않는다. 본 발명의 항체로서, 바람직하게는, 항HER2 항체 (예를 들어, 트라스투주맙 또는 페르투주맙), 항CDH6 항체, 항CD33 항체 또는 항EphA2 항체이며, 보다 바람직하게는, 항HER2 항체이다.Antibodies used in the present invention include, for example, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-CDH6 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD19 antibody. Anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody , anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody or anti-DR5 antibody; , but not limited to these. The antibody of the present invention is preferably an anti-HER2 antibody (eg, Trastuzumab or Pertuzumab), an anti-CDH6 antibody, an anti-CD33 antibody or an anti-EphA2 antibody, and more preferably an anti-HER2 antibody.

본 발명의 항체는, 이 분야에서 통상적으로 실시되는 방법을 사용하여, 항원이 되는 폴리펩티드를 동물에게 면역하고, 생체내에 산생되는 항체를 채취, 정제함으로써 얻을 수 있다. 항원의 유래는 인간에 한정되지 않고, 마우스, 래트 등의 인간 이외의 동물에서 유래하는 항원을 동물에게 면역할 수도 있다. 이 경우에는, 취득된 이종 항원에 결합하는 항체와 인간 항원의 교차성을 시험함으로써, 인간의 질환에 적용 가능한 항체를 선별할 수 있다.The antibody of the present invention can be obtained by immunizing an animal with a polypeptide as an antigen, and collecting and purifying the antibody produced in vivo using a method commonly practiced in this field. The origin of the antigen is not limited to humans, and antigens derived from non-human animals such as mice and rats can be immunized to animals. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between an antibody that binds to the obtained heterologous antigen and a human antigen.

또, 공지된 방법 (예를 들어, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennett, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N. Y. (1980)) 에 따라서, 항원에 대한 항체를 산생하는 항체 산생 세포와 골수종 세포를 융합시킴으로써 하이브리도마를 수립하고, 모노클로날 항체를 얻을 수도 있다.Also, known methods (eg, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennett, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N. Y. (1980)) Accordingly, a monoclonal antibody may be obtained by establishing a hybridoma by fusing an antibody-producing cell that produces an antibody against an antigen with a myeloma cell.

또한, 항원은 항원 단백질을 코드하는 유전자를 유전자 조작에 의해 숙주 세포에 산생시킴으로써 얻을 수 있다.In addition, an antigen can be obtained by producing a gene encoding an antigen protein in a host cell by genetic manipulation.

본 발명의 인간화 항체는 공지된 방법 (예를 들어, Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90/07861) 에 따라서 취득할 수 있다.Humanized antibodies of the present invention can be prepared by known methods (eg, Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90/07861). can be obtained according to

예를 들어, 항HER2 항체 (US5821337, WO2004/008099 등), 항CD33 항체 (WO2014/057687 등), 항CD70 항체 (WO2004/073656 등), 항EphA2 항체 (WO2009/028639 등), 항CDH6 항체 (WO2018/212136 등) 는, 공지된 수단에 의해 취득할 수 있다.For example, anti-HER2 antibodies (US5821337, WO2004/008099, etc.), anti-CD33 antibodies (WO2014/057687, etc.), anti-CD70 antibodies (WO2004/073656, etc.), anti-EphA2 antibodies (WO2009/028639, etc.), anti-CDH6 antibodies ( WO2018/212136 etc.) can be obtained by known means.

본 발명의 항HER2 항체는, 특별히 제한은 없지만, 예를 들어, 이하의 특성을 갖는 것이 바람직하다. The anti-HER2 antibody of the present invention is not particularly limited, but preferably has the following characteristics, for example.

(1) 이하의 특성을 갖는 것을 특징으로 하는 항HER2 항체 ; (1) an anti-HER2 antibody characterized by having the following characteristics;

(a) HER2 에 특이적으로 결합한다.(a) It specifically binds to HER2.

(b) HER2 와 결합함으로써 HER2 발현 세포에 내재화하는 활성을 갖는다.(b) By binding to HER2, it has an activity to internalize into HER2-expressing cells.

(2) HER2 의 세포외 도메인에 결합하는 상기 (1) 에 기재된 항체. (2) The antibody described in (1) above, which binds to the extracellular domain of HER2.

(3) 상기 항체가 모노클로날 항체인 상기 (1) 또는 (2) 에 기재된 항체. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody.

(4) 항체 의존성 세포 상해 (ADCC) 활성 및/또는 보체 의존성 세포 상해 (CDC) 활성을 갖는 상기 (1) ∼ (3) 중 어느 하나에 기재된 항체. (4) The antibody according to any one of (1) to (3) above, having antibody-dependent cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.

(5) 마우스 모노클로날 항체, 키메라 모노크로날 항체 또는 인간화 모노클로날 항체인, 상기 (1) ∼ (4) 중 어느 하나에 기재된 항체. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody.

(6) 중사슬 정상 영역이 인간 IgG1 의 중사슬 정상 영역이며, ADCC 및 ADCP 활성의 저감을 가져오는 변이를 포함하는, 상기 (1) ∼ (3) 에 기재된 항체. (6) The antibody according to (1) to (3) above, wherein the heavy chain constant region is a human IgG1 heavy chain constant region and contains mutations that result in reduction of ADCC and ADCP activities.

(7) 서열 번호 2 에 기재된 아미노산 서열로 이루어지는 중사슬 및 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬을 포함하여 이루어지는 인간화 모노클로날 항체인 상기 (1) ∼ (4) 중 어느 하나에 기재된 항체. (7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 2 and a light chain composed of the amino acid sequence shown in SEQ ID NO: 1 .

(8) 중사슬 정상 영역이 인간 IgG1 의 중사슬 정상 영역이며, EU 인덱스에 의해 나타내는 234 위치 및 235 위치의 류신이 알라닌으로 치환되어 있는, 상기 (5) 에 기재된 항체. (8) The antibody according to (5) above, wherein the heavy chain constant region is a human IgG1 heavy chain constant region, and the leucines at positions 234 and 235 indicated by the EU index are substituted with alanine.

(9) 서열 번호 3 에 기재된 아미노산 서열로 이루어지는 중사슬 및 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬을 포함하여 이루어지는 인간화 모노클로날 항체인 상기 (8) 에 기재된 항체. (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 3 and a light chain composed of the amino acid sequence shown in SEQ ID NO: 1.

(10) 서열 번호 29 에 기재된 아미노산 서열로 이루어지는 중사슬 및 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬을 포함하여 이루어지는 인간화 모노클로날 항체인 상기 (1) ∼ (4) 에 기재된 항체. (10) The antibody according to (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 29 and a light chain composed of the amino acid sequence shown in SEQ ID NO: 28.

(11) 서열 번호 30 에 기재된 아미노산 서열로 이루어지는 중사슬 및 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬을 포함하여 이루어지는 인간화 모노클로날 항체인 상기 (1) ∼ (4) 에 기재된 항체. (11) The antibody according to (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 30 and a light chain composed of the amino acid sequence shown in SEQ ID NO: 28.

(12) 중사슬 카르복실 말단에 있어서 1 또는 2 개의 아미노산이 결실되어 있는 상기 (1) ∼ (11) 중 어느 하나에 기재된 항체. (12) The antibody according to any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

(13) 상기 (1) ∼ (12) 중 어느 하나에 기재된 항체를 코드하는 폴리 뉴클레오티드를 함유하는 발현 벡터에 의해 형질 전환된 숙주 세포를 배양하는 공정 및 당해 공정에서 얻어진 배양물로부터 목적으로 하는 항체를 채취하는 공정을 포함하는 당해 항체의 제조 방법에 의해 얻어지는 항체.(13) A step of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and the antibody of interest from the culture obtained in this step An antibody obtained by a method for producing the antibody comprising a step of collecting.

＜2.2.2 항체의 당사슬 리모델링＞ <2.2.2 Remodeling of antibody chains>

최근, 불균일한 항체의 당사슬을, 효소 반응에 의해 리모델링 하고, 관능기를 갖는 당사슬을 균일하게 도입하는 방법이 보고되어 있다 (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). 이 당사슬 리모델링 기술을 이용하여, 부위 특이적으로 약물을 도입하고, 균일한 ADC 를 합성하는 시도도 이루어지고 있다 (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).Recently, a method of remodeling heterogeneous antibody sugar chains by an enzymatic reaction and uniformly introducing sugar chains having functional groups has been reported (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett 2016, 7, 1005-1008). Attempts to introduce site-specific drugs and synthesize uniform ADCs using this sugar chain remodeling technology have also been made (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

당사슬의 리모델링은 먼저 가수분해 효소를 이용하여, 단백질 (항체 등) 에 부가되어 있는 불균일한 당사슬을 말단의 GlcNAc 만 남기고 절제하고, GlcNAc 가 부가한 균일한 단백질 부분을 조제한다 (이하, 「억셉터」 라고 한다). 다음으로, 별도 조제한 임의의 당사슬을 준비하고 (이하, 「도너」 라고 한다), 이 억셉터와 도너를, 당전이 효소를 사용하여 연결한다. 이에 따라, 임의의 당사슬 구조를 가진 균일한 당단백질을 합성할 수 있다.In the remodeling of sugar chains, first, using a hydrolase, the non-uniform sugar chains added to proteins (antibodies, etc.) are excised leaving only the terminal GlcNAc, and a uniform protein portion to which GlcNAc is added is prepared (hereinafter referred to as "acceptor"). 」). Next, an arbitrary sugar chain prepared separately is prepared (hereinafter referred to as "donor"), and the acceptor and donor are ligated using a glycosyltransferase. Accordingly, a homogeneous glycoprotein having an arbitrary sugar chain structure can be synthesized.

본 발명에 있어서, 「당사슬」 이란, 2 개 이상의 단당이 글리코시드 결합에 의해 결합된 구조 단위를 의미한다. 구체적인 단당이나 당사슬을, 예를 들어 "GlcNAc-", "SG-" 와 같이, 약호로서 표기하는 경우가 있다. 구조식 중에서 이들 약호로 기재한 경우, 환원 말단에서 별도의 구조 단위와의 글리코시드 결합에 귀속하는 산소 원자 또는 질소 원자는, 특별한 정의가 있는 경우를 제외하고, 당해 당사슬을 나타내는 약호에는 포함되지 않는 것으로서 표시된다.In the present invention, "sugar chain" means a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and sugar chains may be expressed as abbreviations, such as "GlcNAc-" and "SG-", for example. When described with these abbreviations in the structural formula, the oxygen atom or nitrogen atom that belongs to the glycosidic bond with another structural unit at the reducing end is not included in the abbreviation representing the sugar chain, unless specifically defined. displayed

본 발명에 있어서, 당사슬의 기본 단위가 되는 단당의 기재는, 별도로 정하는 경우를 제외하고, 편의상, 그 고리 구조에 있어서, 고리를 구성하는 산소 원자에 결합하고, 또한, 하이드록시기 (또는 글리코시드 결합에 귀속하는 산소 원자) 와 직접 결합한 탄소 원자를 1 위치 (시알산에 있어서만 2 위치) 로서 표기한다. 실시예 화합물의 명칭은, 화학 구조 전체적으로 붙여진 것이며, 이 룰은 반드시 적용되는 것은 아니다.In the present invention, the base unit of the monosaccharide serving as the basic unit of the sugar chain is, for convenience, bonded to an oxygen atom constituting the ring in the ring structure, and also has a hydroxyl group (or glycoside), unless otherwise specified. Oxygen atom attributable to bond) and directly bonded carbon atom are expressed as position 1 (position 2 only in sialic acid). The names of the compounds of the examples are assigned to the entire chemical structure, and this rule is not necessarily applied.

본 발명에 있어서, 당사슬을 기호 (예를 들어, SG, MSG, GlcNAc 등) 로서 기재하는 경우, 별도로 정의되는 경우를 제외하고, 환원 말단의 탄소까지를, 당해 기호에 포함시키는 것으로 하고, N- 또는 O-글리코시드 결합에 귀속하는 N 또는 O 는, 당해 기호에는 포함되지 않는 것으로 한다.In the present invention, when a sugar chain is described as a symbol (eg, SG, MSG, GlcNAc, etc.), carbon at the reducing end is included in the symbol unless otherwise defined, and N- Alternatively, N or O belonging to an O-glycosidic bond shall not be included in the symbol.

본 발명의 항체 약물 콘쥬게이트는, 다음 식The antibody drug conjugate of the present invention is the following formula

[화학식 79] [Formula 79]

Figure pat00079
Figure pat00079

으로 나타내고, 항체 Ab 또는 그 기능성 단편은, 그 아미노산 잔기 (예를 들어, 시스테인, 리신 등) 의 측사슬로부터 직접 L 에 결합하거나, Ab 의 당사슬 또는 리모델링 된 당사슬로부터 L 에 결합하고 있다., antibody Ab or a functional fragment thereof binds to L directly from the side chain of its amino acid residue (eg, cysteine, lysine, etc.), or binds to L from the Ab sugar chain or remodeled sugar chain.

본 발명에 있어서의 Ab 의 당사슬은, N 결합형 당사슬이나 O 결합형 당사슬이고, 바람직하게는, N 결합형 당사슬이다.The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

N 결합형 당사슬은 N 글리코시드 결합, O 결합형 당사슬은 O 글리코시드 결합에 의해, 항체의 아미노산 측사슬과 결합하고 있다.The N-linked sugar chain is bonded to the amino acid side chain of the antibody by an N glycosidic bond, and the O-linked sugar chain is bonded to an O glycosidic bond.

본 발명의 Ab 는, IgG 이며, 바람직하게는, IgG1, IgG2 또는 IgG4 이다.The Ab of the present invention is IgG, preferably IgG1, IgG2 or IgG4.

IgG 는 그 중사슬의 Fc 영역에 있어서의 297 번째의 아스파라긴 잔기 (이하, 「Asn297 또는 N297」 이라고 한다.) 에 잘 보존된 N 결합형 당사슬을 갖고 있고, 항체 분자의 활성이나 동태 등에 기여하는 것이 알려져 있다 (Eon-Duval, A. et al, Biotechnol. Prog. 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).IgG has a well-conserved N-linked sugar chain at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, and contributes to the activity and dynamics of antibody molecules. It is known (Eon-Duval, A. et al, Biotechnol. Prog. 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

IgG 의 정상 영역에 있어서의 아미노산 서열은 잘 보존되어 있고, Edelman 등의 보고 (Proc. Natl. Acad. Sci. U.S.A., 63, 78-85, (1969)) 에 있어서, 각각의 아미노산이 EU 번호 (EU INDEX) 로 특정되어 있다. 예를 들어, Fc 영역에 있어서 N 결합형 당사슬이 부가하는 Asn297 은, EU 번호에 있어서 297 위치에 상당하는 것이며, 분자의 단편화나 영역 결손에 의해 실제의 아미노산 위치가 변동한 경우이더라도 EU 번호로 표시함으로써 아미노산이 일의적으로 특정된다.The amino acid sequence in the constant region of IgG is well conserved, and in the report of Edelman et al. (Proc. Natl. Acad. Sci. U.S.A., 63, 78-85, (1969)), each amino acid has an EU number ( EU INDEX). For example, Asn297 added by the N-linked sugar chain in the Fc region corresponds to position 297 in the EU number, and even when the actual amino acid position is changed due to molecular fragmentation or region deletion, it is represented by the EU number. By doing so, amino acids are uniquely specified.

아래 그림은 본 발명의 항체 약물 콘쥬게이트가 항체 또는 그 기능성 단편의 상기 N297 당사슬로부터 L 에 결합하고 있는 경우를 나타낸다.The figure below shows the case where the antibody drug conjugate of the present invention binds to L from the N297 sugar chain of the antibody or functional fragment thereof.

[화학식 80][Formula 80]

Figure pat00080
Figure pat00080

또한, 당해 리모델링 된 당사슬을 갖는 항체를 당사슬 리모델링 항체라고 한다.Also, an antibody having the remodeled sugar chain is referred to as a sugar chain remodeling antibody.

SGP(α2,6-SGP) 는, Sialylglycopeptide 의 약칭이며, N 결합형 당펩티드의 대표적인 것이다. SGP 는, 예를 들어, WO2011/027868 에 기재된 방법에 따라서, 계란의 난황으로부터 단리, 정제할 수 있다. 또, SGP 의 정제품이 도쿄 화성 공업 및 후시미 제약소에서 판매되고 있다. 본 명세서에 있어서, SGP 의 당사슬 부분을 SG 라고 표기하고, SG 의 환원 말단의 GlcNAc 가 1 개 결손된 당사슬을 SG(10) 으로 표기한다. SG(10) 은, 예를 들어, 우메가와 등의 보고 (Biochim. Biophys. Acta 2010, 1800, 1203-1209) 를 참조하여, SGP 의 효소적인 가수분해에 의해 조제할 수 있다. 또, SG(10) 도 도쿄 화성 공업 및 후시미 제약소에서 구입할 수 있다.SGP (α2,6-SGP) is an abbreviation for Sialylglycopeptide, and is a typical N-linked glycopeptide. SGP can be isolated and purified from egg yolk according to the method described in WO2011/027868, for example. In addition, purified products of SGP are sold at Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In the present specification, the sugar chain portion of SGP is denoted as SG, and the sugar chain in which one GlcNAc at the reducing end of SG is missing is denoted as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP with reference to the report of Umegawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209), for example. In addition, SG (10) can also be purchased from Tokyo Chemical Industry and Fushimi Pharmaceutical Co., Ltd.

본 명세서에 있어서, SG(10) 의 β-Man 의 분기 사슬의 어느 일방에서만 비환원 말단의 시알산이 결실된 당사슬 구조를 MSG(9) 로 표기하고, 분기 사슬의 1-3 당사슬에만 시알산을 갖는 것을 MSG1, 분기 사슬의 1-6 당사슬에만 시알산을 갖는 것을 MSG2 라고 각각 표기한다.In the present specification, the sugar chain structure in which sialic acid at the non-reducing end is deleted from only one of the branched chains of β-Man of SG (10) is referred to as MSG (9), and sialic acid is added only to 1-3 sugar chains of the branched chain. Those having sialic acid are indicated as MSG1, and those having sialic acid only in the 1-6 sugar chains of the branched chain are indicated as MSG2.

본 발명의 리모델링 된 당사슬은, N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, 또는 N297-(Fuc)MSG1 과 N297-(Fuc)MSG2 의 혼합물이고, 바람직하게는, N297-(Fuc)SG, N297-(Fuc)MSG1 또는, N297-(Fuc)MSG2 이며, 보다 바람직하게는 N297-(Fuc)SG 또는 N297-(Fuc)MSG1 이다.The remodeled sugar chain of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably. , N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

N297-(Fuc)SG 는 이하의 구조식 또는 서열식으로 나타낸다.N297-(Fuc)SG is represented by the following structural formula or sequence formula.

[화학식 81][Formula 81]

Figure pat00081
Figure pat00081

[화학식 82][Formula 82]

Figure pat00082
Figure pat00082

상기 식 중, 파선은 항체의 Asn297 에 결합하고 있는 것을 나타내고, In the above formula, the broken line indicates binding to Asn297 of the antibody;

L(PEG) 는, -(CH2-CH2-O)n5-CH2-CH2-NH- 를 나타내고, 우단의 아미노기가 N297 당사슬의 β-Man 의 분기 사슬의 1-3 사슬측 및 1-6 사슬측의 양방의 비환원 말단의 시알산의 2 위치의 카르복실기와 아미드 결합하고 있는 것을 나타내고, 아스테리스크는, 상기 링커 L, 특히 링커 L 에 있어서의 Lb 의 1,2,3-트리아졸 고리 상의 1 위치 또는 3 위치의 질소 원자와 결합하고 있는 것을 나타내고, L(PEG) represents -(CH 2 -CH 2 -O)n 5 -CH 2 -CH 2 -NH-, and the amino group at the right end is N297 on the 1-3 chain side of the branched chain of β-Man and 1-6 indicates that the carboxyl group at the 2nd position of the sialic acid at both non-reducing ends on the chain side is amide bonded, and the asterisk is the linker L, particularly the 1,2,3- of Lb in the linker L It shows that it is bonded to the nitrogen atom at the 1st or 3rd position on the triazole ring,

여기서, n5 는 2 ∼ 10 의 정수이며, 바람직하게는, 2 ∼ 5 의 정수이다.Here, n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

N297-(Fuc)MSG1 은 이하의 구조식 또는 서열식으로 나타낸다.N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula.

[화학식 83][Formula 83]

Figure pat00083
Figure pat00083

[화학식 84][Formula 84]

Figure pat00084
Figure pat00084

상기 식 중, 파선은 항체의 Asn297 에 결합하고 있는 것을 나타내고, In the above formula, the broken line indicates binding to Asn297 of the antibody;

L(PEG) 는, -(CH2-CH2-O)n5-CH2-CH2-NH- 를 나타내고, 우단의 아미노기가 N297 당사슬의 β-Man 의 분기 사슬의 1-3 사슬측의 비환원 말단의 시알산의 2 위치의 카르복실기와 아미드 결합하고 있는 것을 나타내고, L(PEG) represents -(CH 2 -CH 2 -O)n 5 -CH 2 -CH 2 -NH-, and the amino group at the right end is on the 1-3 chain side of the β-Man branch chain of the N297 sugar chain represents an amide bond with the carboxyl group at position 2 of the non-reducing terminal sialic acid;

아스테리스크는, 상기 링커 L, 특히 링커 L 에 있어서의 Lb 의 1,2,3-트리아졸 고리 상의 1 위치 또는 3 위치의 질소 원자와 결합하고 있는 것을 나타내고, Asterisk represents linker L, particularly bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb in the linker L,

여기서, n5 는 2 ∼ 10 의 정수이며, 바람직하게는, 2 ∼ 5 의 정수이다.Here, n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

N297-(Fuc)MSG2 는 이하의 구조식 또는 서열식으로 나타낸다.N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula.

[화학식 85][Formula 85]

Figure pat00085
Figure pat00085

[화학식 86][Formula 86]

Figure pat00086
Figure pat00086

상기 식 중, 파선은 항체의 Asn297 에 결합하고 있는 것을 나타내고, In the above formula, the broken line indicates binding to Asn297 of the antibody;

L(PEG) 는, -(CH2-CH2-O)n5-CH2-CH2-NH- 를 나타내고, 우단의 아미노기가 N297 당사슬의 β-Man 의 분기 사슬의 1-6 사슬측의 비환원 말단의 시알산의 2 위치의 카르복실기와 아미드 결합하고 있는 것을 나타내고, 아스테리스크는, 상기 링커 L, 특히 링커 L 에 있어서의 Lb 의 1,2,3-트리아졸 고리 상의 1 위치 또는 3 위치의 질소 원자와 결합하고 있는 것을 나타내고, L(PEG) represents -(CH 2 -CH 2 -O)n 5 -CH 2 -CH 2 -NH-, and the amino group at the right end is on the 1-6 chain side of the β-Man branch chain of the N297 sugar chain It indicates that the carboxyl group at position 2 of the non-reducing terminal sialic acid is bonded with an amide bond, and the asterisk represents the linker L, particularly the linker L at position 1 or 3 on the 1,2,3-triazole ring of Lb in the linker L. Indicates that it is bonded to the nitrogen atom at the position,

여기서, n5 는 2 ∼ 10 의 정수이며, 바람직하게는, 2 ∼ 5 의 정수이다.Here, n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

본 발명의 항체 약물 콘쥬게이트에 있어서의 항체의 N297 당사슬이, N297-(Fuc)SG 인 경우, 항체는 2량체이기 때문에, 항체 약물 콘쥬게이트는 4 개의 링커 L 및 4 개의 약물 D 가 결합된 분자 (상기 m2 = 2) 가 된다. When the N297 sugar chain of the antibody in the antibody drug conjugate of the present invention is N297-(Fuc)SG, since the antibody is a dimer, the antibody drug conjugate is a molecule in which 4 linkers L and 4 drugs D are linked. (above m 2 = 2).

본 발명의 항체 약물 콘쥬게이트에 있어서의 항체의 N297 당사슬이, N297-(Fuc)MSG1 혹은 N297-(Fuc)MSG2 또는 그들의 혼합물인 경우, 항체는 2량체이기 때문에, 항체 약물 콘쥬게이트는 2 개의 링커 L 및 2 개의 약물 D 가 결합된 분자 (상기 m2 = 1) 가 된다 (도 1 참조).When the N297 sugar chain of the antibody in the antibody drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, so the antibody drug conjugate is composed of two linkers. L and two drugs D are combined to form a molecule (m 2 =1 above) (see Fig. 1).

N297 당사슬은, 바람직하게는, N297-(Fuc)SG 혹은 N297-(Fuc)MSG1 또는 N297-(Fuc)MSG2 이며, 보다 바람직하게는, N297-(Fuc)SG 이다.The N297 sugar chain is preferably N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG.

본 발명의 항체 약물 콘쥬게이트에 있어서의 항체의 N297 당사슬이 N297-(Fuc)SG 혹은 N297-(Fuc)MSG1 또는 N297-(Fuc)MSG2 인 경우, 균일성이 높은 ADC 를 취득할 수 있다.When the N297 sugar chain of the antibody in the antibody drug conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2, an ADC with high uniformity can be obtained.

＜3. 제조 방법＞ <3. Manufacturing method>

본 발명의 신규 CDN 유도체 및 그 항체 약물 콘쥬게이트 또는 그들의 제조 중간체의 대표적인 제조 방법을 설명한다. 또한, 이하에 있어서, 화합물을 나타내기 위해서, 각 반응식 중에 나타내는 화합물의 번호를 사용한다. 즉, 「식 (1) 의 화합물」, 「화합물 (1)」 등으로 칭한다. 또, 이외의 번호의 화합물에 대해서도 동일하게 기재한다.Representative methods for preparing the novel CDN derivatives of the present invention and antibody drug conjugates thereof or intermediates thereof will be described. In addition, in the following, in order to represent a compound, the number of the compound shown in each reaction formula is used. That is, "compound of formula (1)", "compound (1)", etc. are called. Moreover, it describes similarly about the compound of a number other than that.

하기의 A 법 ∼ E 법에 있어서, 치환기 R1 ∼ R5, L1, L2, W1, W2, Z1 ∼ Z3 은, 상기와 동일한 의미이다. 치환기 Ra, Rc, Re 및 Rg 는, 천연형 α-아미노산의 측사슬을 나타낸다. 예를 들어, 메틸기, 이소프로필기, sec-부틸기, 이소부틸기, 벤질기 등이다. PRO1 은, 제 1 급 알코올의 보호기를 나타낸다. 적합하게는, 4,4'-디메톡시트리틸기, 4-메톡시트리틸기 등이다. PRO2, PRO3, PRO7, PRO8 은, 제 2 급 알코올의 보호기를 나타낸다. 적합하게는 tert-부틸디메틸실릴기, 트리이소프로필실릴옥시메틸기, 벤조일기, 2-니트로벤질기, 4-메톡시테트라하이드로피란-4-일기 등이다. PRO6 은 카르복실산의 보호기를 나타낸다. 적합하게는, tert-부틸기, 벤질기 등이다. PRO5, PRO9 는, 아민의 보호기를 나타낸다. PRO5 는, 적합하게는, tert-부틸옥시카르보닐기, 9-플루오레닐메틸옥시카르보닐기, 알릴옥시카르보닐기, 2,2,2-트리클로로에톡시카르보닐기, 벤질옥시카르보닐기 등이며, PRO9 는, 적합하게는, 9-플루오레닐메틸옥시카르보닐기 또는 2-(트리메틸실릴)에톡시카르보닐기이다. PRO4 는, 알코올 또는 아민의 보호기를 나타낸다. 알코올의 경우, 적합하게는 tert-부틸디메틸실릴기, 벤조일기 등이며, 아민의 경우, 적합하게는 2-(트리메틸실릴)에톡시카르보닐기, 알릴옥시카르보닐기, tert-부틸옥시카르보닐기 등이다. Qa 는 산소 원자 또는 황 원자를 나타내고, Qb 는 하이드록시기 또는 티올기를 나타낸다. Qa' 및 Qb' 는, 각각 독립적으로, 부 (負) 로 하전한 산소 원자 (O-) 또는 황 원자 (S-) 를 나타낸다. Rx 및 Ry 는, 각각 독립적으로, 할로겐 원자 또는 -O-PRO2 를 나타낸다. n 은 1 내지 3 의 정수를 나타낸다.In methods A to E described below, the substituents R 1 to R 5 , L 1 , L 2 , W 1 , W 2 , and Z 1 to Z 3 have the same meaning as described above. Substituents R a , R c , R e and R g represent side chains of native α-amino acids. For example, methyl group, isopropyl group, sec-butyl group, isobutyl group, benzyl group and the like. PRO 1 represents a primary alcohol protecting group. Preferably, it is a 4,4'-dimethoxytrityl group, 4-methoxytrityl group, etc. PRO 2 , PRO 3 , PRO 7 , and PRO 8 represent a protecting group of a secondary alcohol. Preferable examples thereof include a tert-butyldimethylsilyl group, a triisopropylsilyloxymethyl group, a benzoyl group, a 2-nitrobenzyl group, and a 4-methoxytetrahydropyran-4-yl group. PRO 6 represents a protecting group of carboxylic acid. Suitably, it is a tert-butyl group, a benzyl group, etc. PRO 5 and PRO 9 represent an amine protecting group. PRO 5 is preferably a tert-butyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, benzyloxycarbonyl group or the like, and PRO 9 is suitable Preferably, it is a 9-fluorenylmethyloxycarbonyl group or a 2-(trimethylsilyl)ethoxycarbonyl group. PRO 4 represents a protecting group of alcohol or amine. In the case of an alcohol, it is preferably a tert-butyldimethylsilyl group, a benzoyl group, or the like, and in the case of an amine, it is preferably a 2-(trimethylsilyl)ethoxycarbonyl group, an allyloxycarbonyl group, a tert-butyloxycarbonyl group or the like. Q a represents an oxygen atom or a sulfur atom, and Q b represents a hydroxy group or a thiol group. Q a' and Q b' each independently represent a negatively charged oxygen atom (O - ) or sulfur atom (S - ). R x and R y each independently represent a halogen atom or -O-PRO 2 . n represents an integer of 1 to 3;

A 법 A method

본 발명의 (1) 로 나타내는 CDN 유도체는, 이하에 기재하는 A 법에 따라서 제조할 수 있다.The CDN derivative represented by (1) of the present invention can be produced according to method A described below.

[화학식 87][Formula 87]

Figure pat00087
Figure pat00087

본 제조법은 일반식 (1) 로 나타내는 화합물을 제조하기 위한 방법이다. 본 제조법의 A-1 공정부터 A-5 공정까지는 원 포트 합성이 가능하지만, 이것은, Gaffney 등의 보고 (Org. Lett. 2010, 12, 3269-3271) 를 참조하여 실시할 수 있다.This production method is a method for producing a compound represented by general formula (1). Although one-pot synthesis is possible from step A-1 to step A-5 of the present manufacturing method, this can be carried out with reference to the report of Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[화학식 88][Formula 88]

Figure pat00088
Figure pat00088

(A-1 공정) (Process A-1)

본 공정은, 식 (1a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 가수분해 반응과 시아노에틸기의 제거를 연속해서 실시함으로써, 식 (2a) 의 화합물을 제조하는 공정이다.This step is a step for producing the compound of the formula (2a) by successively subjecting the compound of the formula (1a) to a hydrolysis reaction and removal of a cyanoethyl group using a known organic chemical method.

화합물 (1a) 를 용매 (아세토니트릴, 테트라하이드로푸란, N,N-디메틸포름아미드 또는 그들의 혼합 용매) 중에서, -10 ℃ 부터 반응에 사용하는 용매의 비점까지, 바람직하게는 15 ℃ 부터 35 ℃ 까지에 있어서, 물 및 산 (피리딘트리플루오로아세트산염, 4,5-디시아노이미다졸, 1H-테트라졸 등) 으로 처리함으로써 가수분해 반응을 실시하였다. 화합물 (1a) 1 몰에 대하여, 물은 2 몰 내지 과잉 몰, 바람직하게는 2 몰 내지 10 몰 사용하고, 산은 1 몰 내지 과잉 몰, 바람직하게는 1 몰 내지 5 몰 사용하였다. 반응 시간은 1 분간 내지 3 시간, 바람직하게는 5 분간 내지 30 분간이다. 이어서, 이 반응액에 염기 (tert-부틸아민 등) 를 첨가하여 시아노에틸기를 제거하였다. 염기는, 화합물 (1a) 1 몰에 대하여 과잉 몰, 바람직하게는 30 몰 내지 50 몰 사용하였다. 반응 시간은, 5 분간 내지 6 시간, 바람직하게는 15 분간 내지 1 시간이다. 반응액을 감압하에서 농축하여, 화합물 (2a) 의 미정제체를 얻었다. 화합물 (2a) 의 미정제체는, 정제하지 않고 다음의 공정으로 진행할 수 있다.Compound (1a) is incubated in a solvent (acetonitrile, tetrahydrofuran, N,N-dimethylformamide or a mixed solvent thereof) from -10°C to the boiling point of the solvent used for the reaction, preferably from 15°C to 35°C. , a hydrolysis reaction was carried out by treating with water and an acid (pyridine trifluoroacetate, 4,5-dicyanoimidazole, 1H-tetrazole, etc.). For 1 mole of compound (1a), 2 moles to excess moles of water, preferably 2 moles to 10 moles, and 1 mole to excess moles of acid were used, preferably 1 to 5 moles, based on 1 mole of compound (1a). The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Subsequently, a base (tert-butylamine or the like) was added to the reaction mixture to remove the cyanoethyl group. The base was used in an excess mole, preferably 30 to 50 moles, based on 1 mole of compound (1a). The reaction time is 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution was concentrated under reduced pressure to obtain a crude compound (2a). The crude form of compound (2a) can proceed to the next step without purification.

(A-2 공정) (Process A-2)

본 공정은, 식 (2a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 하이드록시기의 보호기를 제거하고, 식 (3a) 의 화합물을 제조하는 공정이다. 본 공정의 반응을 개시하기 전에, 필요에 따라, 아세토니트릴을 사용하여 1 회 내지 3 회 공비하고, 식 (2a) 의 미정제체를 건조시켰다.This step is a step of preparing the compound of formula (3a) by removing the protective group of the hydroxyl group using a known organic chemical method for the compound of formula (2a). Before starting the reaction of this step, azeotrope was carried out 1 to 3 times using acetonitrile as needed, and the crude product of Formula (2a) was dried.

PRO1 이 4,4'-디메톡시트리틸기인 경우, 화합물 (2a) 를 용매 (디클로로메탄, 클로로포름, 디클로로에탄 등) 중에서, -10 ℃ 부터 반응에 사용하는 용매의 비점까지, 바람직하게는 15 ℃ 부터 35 ℃ 까지에 있어서, 물과 산 (디클로로아세트산, 트리플루오로아세트산 등) 으로 처리함으로써 4,4'-디메톡시트리틸기를 제거하였다. 화합물 (2a) 1 몰에 대하여, 물은 과잉 몰, 바람직하게는 10 몰 내지 20 몰 사용하고, 산은, 반응에 사용하는 용매로 1 ％ 내지 50 ％ (v/v), 바람직하게는 5 ％ 에서 10 ％ (v/v) 까지 희석하고, 그 희석 용액을 과잉 몰, 바람직하게는 5 몰 내지 15 몰 사용하였다. 반응 시간은 1 분간 내지 3 시간, 바람직하게는 5 분간 내지 30 분간이다. 반응액에 피리딘을 첨가하여 반응 정지 처리를 하였다. 피리딘은 사용한 산을 충분히 중화할 수 있는 양, 바람직하게는 산 1 몰에 대하여 2 몰 내지 10 몰 사용하였다. 반응액을 감압하에서 농축하여, 화합물 (3a) 의 미정제체를 얻었다. 화합물 (3a) 의 미정제체를, 탈수 아세토니트릴을 사용하여 3 회 내지 5 회 공비하였다. 마지막 공비 시에 아세토니트릴을 남기고, 화합물 (3a) 의 0.01 M 내지 1 M 의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액을, 그대로 다음의 공정으로 진행하였다.When PRO 1 is a 4,4'-dimethoxytrityl group, compound (2a) is dissolved in a solvent (dichloromethane, chloroform, dichloroethane, etc.) from -10°C to the boiling point of the solvent used for the reaction, preferably 15 From °C to 35 °C, the 4,4'-dimethoxytrityl group was removed by treatment with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.). An excess mole of water, preferably 10 to 20 moles, is used relative to 1 mole of compound (2a), and 1% to 50% (v/v) of acid as a solvent used for the reaction, preferably 5%, is used. It was diluted to 10% (v/v), and an excess mole of the diluted solution, preferably 5 to 15 moles, was used. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. The reaction was terminated by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to neutralize the acid used, preferably 2 to 10 moles per mole of the acid. The reaction solution was concentrated under reduced pressure to obtain a crude compound (3a). The crude compound (3a) was azeotroped 3 to 5 times using dehydrated acetonitrile. At the time of the final azeotrope, acetonitrile was left behind to obtain a 0.01 M to 1 M acetonitrile solution of compound (3a). The obtained acetonitrile solution was proceeded to the next step as it was.

(A-3 공정) (Process A-3)

본 공정은, 식 (3a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (4a) 의 화합물과의 커플링 반응 및 얻어진 커플링체의 황화 반응을 연속해서 실시함으로써, 식 (5a) 의 화합물을 제조하는 공정이다. In this step, the compound of formula (5a) is obtained by successively performing a coupling reaction with a compound of formula (4a) and a sulfurization reaction of the obtained coupling body using a known organic chemical method for the compound of formula (3a). is the process of manufacturing

본 공정의 반응을 개시하기 전에, 화합물 (4a) 를, 탈수 아세토니트릴을 사용하여 3 회 내지 5 회 공비하였다. 마지막 공비 시에 아세토니트릴을 남기고, 화합물 (4a) 의 0.01 M 내지 1 M 의 아세토니트릴 용액을 조제하였다. 이 용액에 건조제 (분말상 또는 펠릿상의 몰레큘러 시브스 3A 혹은 몰레큘러 시브스 4A) 를 첨가하고, 용액을 사용할 때까지 질소 또는 아르곤 분위기하에서 보존하였다.Before starting the reaction in this step, compound (4a) was azeotroped 3 to 5 times using dehydrated acetonitrile. At the time of the last azeotrope, leaving acetonitrile, a 0.01 M to 1 M acetonitrile solution of compound (4a) was prepared. A desiccant (powdered or pelletized Molecular Sieves 3A or Molecular Sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use.

화합물 (3a) 의 아세토니트릴 용액에, 5 ℃ 부터 35 ℃ 까지에 있어서, 공비에 의해 건조시킨 화합물 (4a) 의 아세토니트릴 용액을 첨가함으로써 커플링 반응을 실시하였다. 반응 시간은, 1 분간 내지 24 시간, 바람직하게는 5 분간 내지 6 시간이다. 이어서, 이 반응액에 황화제 (N,N-디메틸-N'-(3-술파닐리덴-3H-1,2,4-디티아졸-5-일)메탄이미드아미드, 3H-1,2-벤조디티올-3-온 등) 첨가하여 황화 반응을 실시하였다. 황화제는 화합물 (3a) 1 몰에 대하여 1 몰 내지 5 몰, 바람직하게는 1 몰 내지 2 몰 사용하였다. 반응 시간은 5 분간 내지 24 시간, 바람직하게는 30 분간 내지 6 시간이다. 반응액을 감압하에서 농축하여, 화합물 (5a) 의 미정제체를 얻었다. 얻어진 화합물 (5a) 의 미정제체는, 그대로 다음의 공정으로 진행하였다.A coupling reaction was performed by adding an azeotropically dried acetonitrile solution of compound (4a) to an acetonitrile solution of compound (3a) at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Subsequently, a sulfurizing agent (N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, 3H-1,2 was added to the reaction solution. -benzodithiol-3-one, etc.) was added to carry out a sulfurization reaction. The sulfurizing agent was used in an amount of 1 to 5 moles, preferably 1 to 2 moles, based on 1 mole of the compound (3a). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure to obtain a crude compound (5a). The obtained crude compound (5a) proceeded to the next step as it was.

(A-4 공정) (Process A-4)

본 공정은, 식 (5a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 하이드록시기의 보호기를 제거하고, 식 (6a) 의 화합물을 제조하는 공정이다.This step is a step of preparing the compound of formula (6a) by removing the protective group of the hydroxyl group from the compound of formula (5a) using a known organic chemical method.

PRO1 이 4,4'-디메톡시트리틸기인 경우, 화합물 (5a) 의 화합물을 용매 (디클로로메탄, 클로로포름, 디클로로에탄 등) 중에서, -10 ℃ 부터 반응에 사용하는 용매의 비점까지, 바람직하게는 15 ℃ 부터 35 ℃ 까지에 있어서, 물과 산 (디클로로아세트산, 트리플루오로아세트산 등) 으로 처리함으로써 4,4'-디메톡시트리틸기를 제거하였다. 화합물 (5a) 1 몰에 대하여, 물은 과잉 몰, 바람직하게는 10 내지 20 몰을 사용하고, 산은, 반응에 사용하는 용매로 1 ％ 내지 50 ％ (v/v), 바람직하게는 5 ％ 에서 10 ％ (v/v) 까지 희석하고, 그 희석 용액을 과잉 몰, 바람직하게는 5 몰 내지 15 몰 사용하였다. 반응 시간은 1 분간 내지 3 시간, 바람직하게는 5 분간 내지 30 분간이다. 반응액에 피리딘을 첨가하여 반응 정지 처리를 하였다. 피리딘은 사용한 산을 충분히 중화할 수 있는 양, 바람직하게는 산 1 몰에 대하여 10 몰 내지 200 몰 사용하였다. 반응액을 감압하에서 농축하여, 화합물 (6a) 의 미정제체를 얻었다. 얻어진 화합물 (6a) 의 미정제체를, 그대로 다음의 공정으로 진행하였다.When PRO 1 is a 4,4'-dimethoxytrityl group, the compound of compound (5a) is dissolved in a solvent (dichloromethane, chloroform, dichloroethane, etc.) from -10°C to the boiling point of the solvent used for the reaction, preferably 4,4'-dimethoxytrityl group was removed by treatment with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) at 15 °C to 35 °C. An excess mole of water, preferably 10 to 20 moles, is used for 1 mole of compound (5a), and 1% to 50% (v/v) of acid is used as a solvent for the reaction, preferably 5%. It was diluted to 10% (v/v), and an excess mole of the diluted solution, preferably 5 to 15 moles, was used. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. The reaction was terminated by adding pyridine to the reaction solution. Pyridine was used in an amount capable of sufficiently neutralizing the acid used, preferably 10 to 200 moles per mole of the acid. The reaction solution was concentrated under reduced pressure to obtain a crude compound (6a). The obtained crude compound (6a) was proceeded as it was to the next step.

(A-5 공정) (Process A-5)

본 공정은, 식 (6a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 고리화 반응과 황화 반응을 연속해서 실시하고, 식 (7a) 의 화합물을 제조하는 공정이다. This step is a step in which a compound of formula (6a) is continuously subjected to a cyclization reaction and a sulfurization reaction using a known organic chemical method to produce a compound of formula (7a).

화합물 (6a) 를 피리딘에 용해 후, 감압하에서 농축하여 0.01 M 내지 0.5 M의 피리딘 용액을 조제하였다. 이 피리딘 용액에, 5 ℃ 부터 35 ℃ 까지에 있어서, 탈수 축합제 (2-클로로-5,5-디메틸-1,3,2λ5-디옥사포스피난-2-온 등) 를 첨가함으로써 고리화 반응을 실시하였다. 탈수 축합제는, 화합물 (6a) 1 몰에 대하여, 1 몰 내지 과잉 몰, 바람직하게는 3 몰 내지 5 몰 사용하였다. 반응 시간은 1 분간 내지 6 시간, 바람직하게는 5 분간 내지 1 시간이다. 이어서, 이 반응액에 물과 황화제 (3H-1,2-벤조디티올-3-온, N,N-디메틸-N'-(3-술파닐리덴-3H-1,2,4-디티아졸-5-일)메탄이미드아미드 등) 를 첨가하여 황화 반응을 실시하였다. 화합물 (6a) 1 몰에 대하여, 물은 과잉 몰, 바람직하게는 30 몰 내지 50 몰 사용하고, 황화제는 1 몰 내지 5 몰, 바람직하게는 1 몰 내지 2 몰 사용하였다. 반응 시간은 5 분간 내지 12 시간, 바람직하게는 30 분간 내지 3 시간이다. 반응액을 탄산수소나트륨 수용액 (0.1 M 부터 1 M 까지) 에 첨가한 후, 15 분간 내지 24 시간 교반하여 반응 정지 처리를 하였다. 반응액을 유기 용매 (아세트산에틸, 디에틸에테르, 톨루엔 또는 그들의 혼합 용매) 로 1 회 내지 5 회 추출한 후, 추출액을 아울러 무수 염 (무수 황산나트륨 또는 무수 황산마그네슘) 으로 건조시켰다. 건조제를 여과 제거하고 여과액을 감압하에서 농축하였다. 얻어진 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올, 아세트산에틸/메탄올, 헥산/아세트산에틸 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴] 또는 그것들을 조합함으로써 정제하고, 화합물 (7a) 를 2 종류 이상의 디아스테레오머 혼합물 또는 2 종류 이상의 순수한 디아스테레오머로서 얻었다. 본 공정에서는 많은 경우, 2 종류의 디아스테레오머가 얻어지지만, 원료 (1a) 및 (4a) 에 의존하여, 추가로 1 종류 또는 2 종류의 디아스테레오머가 얻어지는 경우도 있다. 얻어진 화합물 (7a) 가 복수의 디아스테레오머 혼합물이더라도, 그 이상의 정제를 하지 않고 다음의 공정으로 진행할 수 있다.After dissolving compound (6a) in pyridine, it was concentrated under reduced pressure to prepare a 0.01 M to 0.5 M pyridine solution. Cyclization is performed by adding a dehydrating condensing agent (2-chloro-5,5-dimethyl-1,3,2λ 5 -dioxaphosphinan-2-one or the like) to this pyridine solution at 5°C to 35°C. reaction was carried out. The dehydration and condensation agent was used in an amount of 1 mol to excess mol, preferably 3 mol to 5 mol, based on 1 mol of compound (6a). The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Subsequently, water and a sulfurizing agent (3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithio Azol-5-yl)methanimidamide, etc.) was added to carry out a sulfurization reaction. An excess mole of water, preferably 30 to 50 moles, and 1 to 5 moles of sulfurizing agent, preferably 1 to 2 moles, were used for 1 mole of compound (6a). The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. After adding the reaction solution to an aqueous solution of sodium hydrogen carbonate (from 0.1 M to 1 M), the reaction was stopped by stirring for 15 minutes to 24 hours. The reaction solution was extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene or a mixed solvent thereof), and then the extract was dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was filtered off and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [dichloromethane/methanol, ethyl acetate/methanol, hexane/ethyl acetate, etc.], C18 silica gel column chromatography [buffer/acetonitrile] or a combination thereof, and the compound (7a ) was obtained as a mixture of two or more diastereomers or two or more pure diastereomers. In this step, in many cases, two types of diastereomers are obtained, but depending on the raw materials (1a) and (4a), one or two types of diastereomers may be further obtained in some cases. Even if the obtained compound (7a) is a mixture of a plurality of diastereomers, it can proceed to the next step without further purification.

(A-6 공정) (Process A-6)

본 공정은, 식 (7a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 시아노에틸기 및 모든 아실계 보호기를 동시에 제거하고, 식 (8a) 의 화합물을 제조하는 공정이다. 본 공정은, 필요에 따라 오토클레이브 안, 또는 실드 튜브 안에서 실시하였다.This step is a step for preparing the compound of formula (8a) by simultaneously removing the cyanoethyl group and all acyl protecting groups from the compound of formula (7a) using a known organic chemical method. This process was carried out in an autoclave or in a sealed tube as needed.

PRO4 가 벤조일기인 경우, 화합물 (7a) 의 화합물을 용매 (메탄올, 에탄올, 테트라하이드로푸란 또는 그들의 혼합 용매) 중에서, 5 ℃ 부터 반응에 사용하는 용매의 비점까지에 있어서, 28 ％ (v/v) 암모니아수로 처리함으로써 시아노에틸기와 벤조일기를 제거하였다. 암모니아는, 화합물 (7a) 1 몰에 대하여 과잉 몰, 바람직하게는 300 몰 내지 3000 몰 사용하였다. 반응 시간은 30 분간 내지 96 시간, 바람직하게는 2 시간 내지 48 시간이다. 필요에 따라 반응액을 농축하고, 잔류물을 분취 HPLC [완충액/아세토니트릴, 완충액/메탄올 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴, 완충액/메탄올 등] 또는 그것들을 조합함으로써 정제하고, 화합물 (8a) 를 얻었다. 얻어진 화합물 (8a) 가 디아스테레오머 혼합물이더라도, 그 이상의 정제를 하지 않고 다음의 공정으로 진행할 수 있다. 또, 본 공정에서는 정제를 하지 않고 그대로 다음의 공정으로 진행할 수도 있다.When PRO 4 is a benzoyl group, the compound of compound (7a) is dissolved in a solvent (methanol, ethanol, tetrahydrofuran or a mixed solvent thereof) from 5°C to the boiling point of the solvent used for the reaction, 28% (v/v ) Cyanoethyl group and benzoyl group were removed by treatment with aqueous ammonia. An excess amount of ammonia, preferably 300 to 3000 moles, was used relative to 1 mole of compound (7a). The reaction time is 30 minutes to 96 hours, preferably 2 hours to 48 hours. The reaction solution was concentrated as necessary, and the residue was purified by preparative HPLC [buffer/acetonitrile, buffer/methanol, etc.], C18 silica gel column chromatography [buffer/acetonitrile, buffer/methanol, etc.] or a combination thereof, , to obtain compound (8a). Even if the obtained compound (8a) is a diastereomer mixture, it can proceed to the next step without further purification. In addition, in this process, it may proceed to the next process as it is without purification.

(A-7 공정) (Process A-7)

본 공정은, 식 (8a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 모든 실릴계 보호기를 동시에 제거하고, 식 (9a) 의 화합물을 제조하는 공정이다. This step is a step for preparing a compound of formula (9a) by simultaneously removing all silyl-based protecting groups from the compound of formula (8a) using a known organic chemical method.

PRO2 및 PRO3 이 tert-부틸디메틸실릴기인 경우, 화합물 (8a) 를, 5 ℃ 부터 100 ℃ 까지, 바람직하게는 35 ℃ 부터 60 ℃ 까지에 있어서, 직접 트리에틸아민3불화수소산염으로 처리함으로써 tert-부틸디메틸실릴기를 제거하였다. 트리에틸아민3불화수소산염은, 화합물 (8a) 1 몰에 대하여 과잉 몰, 바람직하게는 100 내지 200 몰 사용하였다. 반응 시간은 30 분간 내지 24 시간, 바람직하게는 2 시간 내지 12 시간이다. 반응액을 실온까지 냉각 후, 반응액에 빙랭한 1 M 탄산수소트리에틸암모늄 수용액과 트리에틸아민의 3 : 1 내지 10 : 1 (v/v) 의 혼합 용액을 조금씩 따라 반응 정지 처리를 하였다. 필요에 따라, 반응액을 빙랭한 1 M 탄산수소트리에틸암모늄 수용액과 트리에틸아민의 혼합 용액에 따라도 된다. 이 경우에는, 반응 용기를 아세토니트릴과 물로 세정하였다. 트리에틸아민은 반응액의 액성을 약염기성으로 변화시킬 수 있는 충분량, 바람직하게는 트리에틸아민3불화수소산염 1 몰에 대하여, 트리에틸아민을 약 2 몰 사용한다. 반응액의 유기 용매 성분을 감압하에서 증류 제거한 후, 잔류한 수용액을 분취 HPLC [완충액/아세토니트릴, 완충액/메탄올 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴, 완충액/메탄올 등] 또는 그것들을 조합함으로써 정제하고, 화합물 (9a) 를 단일의 디아스테레오머로서 얻었다.When PRO 2 and PRO 3 are tert-butyldimethylsilyl groups, compound (8a) is directly treated with triethylamine hydrofluoric acid at 5°C to 100°C, preferably 35°C to 60°C, thereby The tert-butyldimethylsilyl group was removed. Triethylamine hydrofluoric acid salt was used in an excess mole, preferably 100 to 200 moles, per mole of compound (8a). The reaction time is 30 minutes to 24 hours, preferably 2 hours to 12 hours. After cooling the reaction solution to room temperature, an ice-cooled 3:1 to 10:1 (v/v) mixed solution of 1 M aqueous solution of triethylammonium hydrogen carbonate and triethylamine was added little by little to the reaction solution to stop the reaction. If necessary, an ice-cooled 1 M aqueous solution of triethylammonium hydrogen carbonate and triethylamine may be used as a mixed solution. In this case, the reaction vessel was washed with acetonitrile and water. Triethylamine is used in a sufficient amount to change the liquidity of the reaction solution to weak basicity, preferably about 2 moles of triethylamine per 1 mole of triethylamine trifluoroacetate. After the organic solvent component of the reaction solution was distilled off under reduced pressure, the remaining aqueous solution was preparative HPLC [buffer/acetonitrile, buffer/methanol, etc.], C18 silica gel column chromatography [buffer/acetonitrile, buffer/methanol, etc.] or those were purified by combining them to obtain compound (9a) as a single diastereomer.

(A-8 공정) (Process A-8)

본 공정은, 식 (9a) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 이온 교환하고, 식 (1) 의 화합물을 제조하는 공정이다. This step is a step of producing a compound of formula (1) by ion-exchanging the compound of formula (9a) using a known organic chemical method.

양이온 교환 수지 (BT AG (등록상표) 50W-X2 레진, 100-200 메시, 수소형) 를 순수에 현탁시키고, 빈 칼럼 카트리지에 충전하였다. 양이온 교환 수지는, 중량비로 화합물 (9a) 의 10 배 내지 50 배량 사용하였다. 과잉의 순수를 자연 유하시킨 후, 1 M 수산화나트륨 수용액을 3 칼럼 체적 자연 유하시키고, 이어서, 6 칼럼 체적의 순수를 자연 유하시켰다. 화합물 (9a) 를 약 3 칼럼 체적의 순수에 용해하여, 칼럼에 차지하였다. 화합물이 순수에 잘 용해되지 않는 경우에는, 소량의 유기 용매 (아세토니트릴, 메탄올 등) 와의 혼합액을 사용해도 된다. 자연 유하한 용액을 분취 후, 추가로 6 칼럼 체적의 순수 등으로 용출하고, 획분을 분취하였다. 목적물을 포함하는 획분을 아울러 동결 건조시켜, 화합물 (1) 을 단일의 디아스테레오머로서 얻었다.A cation exchange resin (BT AG (registered trademark) 50W-X2 resin, 100-200 mesh, hydrogen type) was suspended in pure water and charged into an empty column cartridge. The cation exchange resin was used in an amount of 10 to 50 times that of Compound (9a) by weight ratio. After the excess pure water was allowed to flow naturally, a 1 M sodium hydroxide aqueous solution was allowed to flow naturally in 3 column volumes, and then the pure water in the volume of 6 columns was naturally flowed down. Compound (9a) was dissolved in about 3 column volumes of pure water and charged into the column. When the compound does not dissolve well in pure water, a liquid mixture with a small amount of an organic solvent (acetonitrile, methanol, etc.) may be used. After fractionation of the naturally flowing solution, it was further eluted with 6 column volumes of pure water or the like, and fractions were fractionated. Fractions containing the target substance were collectively lyophilized to obtain compound (1) as a single diastereomer.

A' 법 A' Law

본 발명의 (1') 로 나타내는 CDN 유도체는, 이하에 기재하는 A' 법에 따라서 제조할 수 있다.The CDN derivative represented by (1') of the present invention can be produced according to method A' described below.

[화학식 89][Formula 89]

Figure pat00089
Figure pat00089

본 제조법은, A 법의 일부를 변경하여 일반식 (1') 로 나타내는 화합물을 제조하기 위한 방법이다. 구체적으로는, 일반식 (1') 의 화합물은 A 법 A-5 공정을 이하에 나타내는 A'-5 공정으로 변경하여 제조할 수 있다. 또, 치환기 Rx 와 Ry 가 양방 할로겐 원자인 경우에는, A-7 공정을 생략할 수 있다.This production method is a method for producing a compound represented by general formula (1') by changing a part of Method A. Specifically, the compound of the general formula (1') can be produced by changing method A, step A-5 to step A'-5 shown below. In addition, when the substituents R x and R y are both halogen atoms, the step A-7 can be omitted.

[화학식 90][Formula 90]

Figure pat00090
Figure pat00090

(A'-5 공정) (A'-5 process)

본 공정은, 식 (6a') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 고리화 반응과 산화 반응을 연속해서 실시하고, 식 (7a') 의 화합물을 제조하는 공정이다. This step is a step in which a compound of formula (6a') is continuously subjected to a cyclization reaction and an oxidation reaction using a known organic chemical method to produce a compound of formula (7a').

화합물 (6a') 를 피리딘에 용해 후, 감압하에서 농축하여 0.01 M 내지 0.5 M의 피리딘 용액을 조제하였다. 이 피리딘 용액에, 5 ℃ 부터 35 ℃ 까지에 있어서, 탈수 축합제 (2-클로로-5,5-디메틸-1,3,2λ5-디옥사포스피난-2-온 등) 를 첨가하여 고리화 반응을 실시하였다. 탈수 축합제는, 화합물 (6a') 1 몰에 대하여, 1 몰 내지 과잉 몰, 바람직하게는 3 몰 내지 5 몰 사용하였다. 반응 시간은 1 분간 내지 6 시간, 바람직하게는 5 분간 내지 1 시간이다. 이어서, 이 반응액에 물과 산화제 (요오드 등) 를 첨가하여 산화 반응을 실시하였다. 화합물 (6a') 1 몰에 대하여, 물은 0 몰 내지 과잉 몰, 바람직하게는 30 몰 내지 50 몰 사용하고, 산화제는 2 몰 내지 10 몰, 바람직하게는 3 몰 내지 5 몰 사용하였다. 반응 시간은 5 분간 내지 12 시간, 바람직하게는 30 분간 내지 3 시간이다. 반응액을 탄산수소나트륨 수용액 (0.1 M 부터 1 M 까지) 에 첨가하고, 15 분간 내지 24 시간 교반하여 반응 정지 처리를 하였다. 반응액을 유기 용매 (아세트산에틸, 디에틸에테르, 톨루엔 또는 그들의 혼합 용매) 로 1 회 내지 5 회 추출한 후, 추출액을 아울러 무수 염 (무수 황산나트륨 또는 무수 황산마그네슘) 으로 건조시켰다. 건조제를 여과 제거하고 여과액을 감압하에서 농축하였다. 얻어진 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올, 아세트산에틸/메탄올, 헥산/아세트산에틸 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴] 또는 그것들을 조합함으로써 정제하고, 화합물 (7a') 를 얻었다.After dissolving compound (6a') in pyridine, it was concentrated under reduced pressure to prepare a 0.01 M to 0.5 M pyridine solution. To this pyridine solution, a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ 5 -dioxaphosphinan-2-one or the like) is added at 5°C to 35°C for cyclization reaction was carried out. The dehydration and condensation agent was used in an amount of 1 mol to excess mol, preferably 3 mol to 5 mol, based on 1 mol of compound (6a'). The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Subsequently, an oxidation reaction was performed by adding water and an oxidizing agent (such as iodine) to the reaction solution. 0 to excess mole, preferably 30 to 50 moles of water, and 2 to 10 moles, preferably 3 to 5 moles of oxidizing agent, were used for 1 mole of compound (6a'). The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction solution was added to an aqueous sodium hydrogencarbonate solution (0.1 M to 1 M), and the reaction was stopped by stirring for 15 minutes to 24 hours. The reaction solution was extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene or a mixed solvent thereof), and then the extract was dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was filtered off and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [dichloromethane/methanol, ethyl acetate/methanol, hexane/ethyl acetate, etc.], C18 silica gel column chromatography [buffer/acetonitrile] or a combination thereof, and the compound (7a ') was obtained.

A'' 법 A'' Law

본 발명의 (1'') 로 나타내는 CDN 유도체는, 이하에 기재하는 A'' 법에 따라서 제조할 수 있다.The CDN derivative represented by (1'') of the present invention can be produced according to the A'' method described below.

[화학식 91][Formula 91]

Figure pat00091
Figure pat00091

본 제조법은, A 법의 일부를 변경하여 일반식 (1'') 로 나타내는 화합물을 제조하기 위한 방법이다. 구체적으로는, 일반식 (1'') 의 화합물은, A 법의 A-3 공정을 이하에 나타내는 A''-3 공정으로 변경하여 제조할 수 있다. 또, 치환기 Rx 와 Ry 가 양방 할로겐 원자인 경우에는, A-7 공정을 생략할 수 있다.This production method is a method for producing a compound represented by general formula (1'') by changing a part of Method A. Specifically, the compound of the general formula (1'') can be produced by changing the step A-3 of method A to the step A''-3 shown below. In addition, when the substituents R x and R y are both halogen atoms, the step A-7 can be omitted.

[화학식 92][Formula 92]

Figure pat00092
Figure pat00092

(A''-3 공정) (A''-3 process)

본 공정은, 식 (3a'') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (4a'') 의 화합물과의 커플링 반응 및 얻어진 커플링체의 산화 반응을 연속해서 실시함으로써, 식 (5a'') 의 화합물을 제조하는 공정이다. In this step, the compound of formula (3a″) is continuously subjected to a coupling reaction with a compound of formula (4a″) using a known organic chemical method and an oxidation reaction of the obtained coupling body, thereby obtaining the formula ( This is a process for preparing the compound of 5a″).

본 공정의 반응을 개시하기 전에, 화합물 (4a'') 를, 탈수 아세토니트릴을 사용하여 3 회 내지 5 회 공비하였다. 마지막 공비 시에 아세토니트릴을 남기고, 화합물 (4a'') 의 0.01 M 내지 1 M 의 아세토니트릴 용액을 조제하였다. 이 용액에 건조제 (분말상 또는 펠릿상의 몰레큘러 시브스 3A 혹은 몰레큘러 시브스 4A) 를 첨가하고, 용액을 사용할 때까지 질소 또는 아르곤 분위기하에서 보존하였다.Before starting the reaction in this step, the compound (4a″) was azeotroped 3 to 5 times using dehydrated acetonitrile. At the time of the last azeotrope, leaving acetonitrile, a 0.01 M to 1 M acetonitrile solution of compound (4a″) was prepared. A desiccant (powdered or pelletized Molecular Sieves 3A or Molecular Sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use.

화합물 (3a'') 의 아세토니트릴 용액에, 5 ℃ 부터 35 ℃ 까지에 있어서, 공비에 의해 건조시킨 화합물 (4a'') 의 아세토니트릴 용액을 첨가함으로써 커플링 반응을 실시하였다. 반응 시간은, 1 분간 내지 24 시간, 바람직하게는 5 분간 내지 6 시간이다. 이어서, 이 반응액에 산화제 (tert-부틸하이드로퍼옥사이드 등) 를 첨가하여 산화 반응을 실시하였다. 산화제는 화합물 (3a'') 1 몰에 대하여 1 몰 내지 5 몰, 바람직하게는 2 몰 내지 3 몰 사용하였다. 반응 시간은 5 분간 내지 24 시간, 바람직하게는 30 분간 내지 6 시간이다. 반응액에 포화 티오황산나트륨 수용액을 첨가하고, 10 분간 내지 12 시간 교반하여 반응 정지 처리를 하였다. 반응액을 유기 용매 (디클로로메탄과 메탄올의 혼합 용매 등) 로 1 회 내지 5 회 추출한 후, 추출액을 아울러 무수 염 (무수 황산나트륨 또는 무수 황산마그네슘) 으로 건조시켰다. 건조제를 여과 제거하고 여과액을 감압하에서 농축하여, 화합물 (5a'') 의 미정제체를 얻었다. 얻어진 화합물 (5a'') 의 미정제체를 그대로 다음의 공정으로 진행하였다.Coupling reaction was performed by adding an azeotropically dried acetonitrile solution of compound (4a″) to an acetonitrile solution of compound (3a″) at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Then, an oxidizing agent (tert-butyl hydroperoxide or the like) was added to this reaction solution to carry out an oxidation reaction. The oxidizing agent was used in an amount of 1 to 5 moles, preferably 2 to 3 moles, based on 1 mole of the compound (3a″). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. A saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and the reaction was stopped by stirring for 10 minutes to 12 hours. After the reaction solution was extracted 1 to 5 times with an organic solvent (such as a mixed solvent of dichloromethane and methanol), the extract was dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude compound (5a″). The obtained crude compound (5a″) was proceeded as it was to the next step.

A''' 법 A''' Law

본 발명의 (1''') 로 나타내는 CDN 유도체는, 이하에 기재하는 A''' 법에 따라서 제조할 수 있다.The CDN derivative represented by (1''') of the present invention can be produced according to the A''' method described below.

[화학식 93][Formula 93]

Figure pat00093
Figure pat00093

본 제조법은, A 법의 일부를 변경하여 일반식 (1''') 로 나타내는 화합물을 제조하기 위한 방법이다. 구체적으로는, 일반식 (1''') 의 화합물은, A 법의 A-3 공정을 A''-3 공정으로, A-5 공정을 A'-5 공정으로 변경하여 제조할 수 있다. 또, 치환기 Rx 와 Ry 가 양방 할로겐 원자인 경우에는, A-7 공정을 생략할 수 있다.This production method is a method for producing a compound represented by the general formula (1''') by changing a part of Method A. Specifically, the compound of the general formula (1''') can be produced by changing step A-3 of method A to step A''-3 and step A-5 to step A'-5. In addition, when the substituents R x and R y are both halogen atoms, the step A-7 can be omitted.

[화학식 94][Formula 94]

Figure pat00094
Figure pat00094

B 법 : 콘쥬게이션 전구체 (당사슬 콘쥬게이션) Method B: Conjugation precursor (sugar chain conjugation)

본 발명의 (2) 로 나타내는 콘쥬게이션 전구체는, 이하에 기재하는 B 법에 따라서 제조할 수 있다.The conjugation precursor represented by (2) of the present invention can be produced according to method B described below.

[화학식 95][Formula 95]

Figure pat00095
Figure pat00095

본 제조법은, L1 의 임의의 위치에 -NH2 가 치환되어 있는 경우의 콘쥬게이션 전구체 (2) 를 제조하기 위한 방법이다.This production method is a method for producing a conjugation precursor (2) in which -NH 2 is substituted at any position of L 1 .

[화학식 96][Formula 96]

Figure pat00096
Figure pat00096

(B-1 공정) (B-1 process)

본 공정은, 식 (1b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 보호기를 제거함으로써, 식 (2b) 의 화합물을 제조하는 공정이다.This step is a step for preparing the compound of the formula (2b) by removing the protecting group from the compound of the formula (1b) using a known organic chemical method.

PRO5 가 tert-부틸옥시카르보닐기인 경우, 화합물 (1b) 를 용매 (디클로로메탄, 디옥산, 아세토니트릴, 아세트산에틸, 테트라하이드로푸란, 또는 그들의 혼합 용매) 중에서, -10 ℃ 부터 반응에 사용하는 용매의 비점까지, 바람직하게는 15 ℃ 부터 35 ℃ 까지에 있어서, 트리플루오로아세트산으로 처리함으로써 보호기를 제거하였다. 트리플루오로아세트산은, 화합물 (1b) 1 몰에 대하여 과잉 몰, 바람직하게는 20 몰 내지 50 몰 사용하였다. 반응 시간은 5 분간 내지 24 시간, 바람직하게는 30 분간 내지 6 시간이다. 반응액을 감압하에서 농축한 후, 톨루엔에 현탁시켜 재차 감압하에서 농축하였다. 이 조작을 2 회 내지 5 회 반복하였다. 용매 (디에틸에테르, 디이소프로필에테르, 헥산, 디클로로메탄, 아세트산에틸 또는 그들의 혼합 용매) 를 첨가하여 슬러리상으로 한 후, 고체를 여과 채취하고, 화합물 (2b) 의 미정제체를 얻었다. 화합물 (2b) 의 미정제체는, 이 이상의 정제를 하지 않고 다음의 공정으로 진행하였다.When PRO 5 is a tert-butyloxycarbonyl group, compound (1b) is dissolved in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixed solvent thereof) from -10°C to the solvent used for the reaction The protecting group was removed by treatment with trifluoroacetic acid, preferably from 15 °C to 35 °C, up to the boiling point of . Trifluoroacetic acid was used in an excess mole, preferably 20 to 50 moles, based on 1 mole of compound (1b). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. After concentrating the reaction solution under reduced pressure, it was suspended in toluene and concentrated again under reduced pressure. This operation was repeated 2 to 5 times. After adding a solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate or a mixed solvent thereof) to form a slurry, the solid was collected by filtration to obtain a crude compound (2b). The crude compound of compound (2b) proceeded to the next step without further purification.

(B-2 공정) (B-2 process)

본 공정은, 식 (2b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (3b) 의 화합물과의 아미드화를 실시함으로써, 식 (4b) 의 화합물을 제조하는 공정이다.This step is a step for producing a compound of formula (4b) by subjecting the compound of formula (2b) to amidation with a compound of formula (3b) using a known organic chemical method.

화합물 (2b) 를 용매 (N,N-디메틸포름아미드, N-메틸피롤리돈, N,N-디메틸아세트아미드, 아세토니트릴 등) 중에서, 5 ℃ 부터 35 ℃ 까지에 있어서, 염기 (트리에틸아민, N,N-디이소프로필에틸아민 등) 및 화합물 (3b) 와 반응시킴으로써 아미드화를 실시하였다. 화합물 (2b) 1 몰에 대하여, 염기는 1 몰 내지 5 몰 사용하고, 화합물 (3b) 는 0.5 몰 내지 1.5 몰 사용하였다. 반응 시간은 10 분간 내지 72 시간, 바람직하게는 1 시간 내지 24 시간이다. 반응액을 유기 용매 (디클로로메탄, 클로로포름, 아세트산에틸, 메탄올 또는 그 혼합 용매) 와 물 또는 산성 수용액 (0.1 에서 1 M 의 염산, 시트르산 수용액 등) 의 2 층에 따르고, 유기 용매로 1 회 내지 5 회 추출하였다. 추출액을 아울러 포화 식염수로 세정 후, 무수 염 (무수 황산나트륨 또는 무수 황산마그네슘) 으로 건조시켰다. 건조제를 여과 제거하고 여과액을 감압하에서 농축하였다. 또한, 상기 분액 조작을 생략하고, 반응액을 그대로 감압하에서 농축하여 다음의 실리카 겔 칼럼 정제로 진행할 수도 있다. 얻어진 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올, 아세트산에틸/메탄올 등] 으로 정제하고, 화합물 (4b) 를 얻었다. 필요에 따라, 얻어진 화합물 (4b) 를 양용매 (아세트산에틸, 아세토니트릴, 디클로로메탄, 메탄올 또는 그들의 혼합 용매) 에 용해한 후, 빈용매 (디에틸에테르, 디이소프로필에테르, 헥산 등) 를 첨가하여 재침전하고, 고체를 여과 채취함으로써 순도를 올릴 수 있다.Compound (2b) is dissolved in a solvent (N,N-dimethylformamide, N-methylpyrrolidone, N,N-dimethylacetamide, acetonitrile, etc.) at 5°C to 35°C in a base (triethylamine) , N,N-diisopropylethylamine, etc.) and compound (3b) to carry out amidation. Based on 1 mole of compound (2b), 1 to 5 moles of the base were used, and 0.5 to 1.5 moles of compound (3b) were used. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution was poured into two layers of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol or a mixed solvent thereof) and water or an acidic aqueous solution (0.1 to 1 M hydrochloric acid, citric acid aqueous solution, etc.), 1 to 5 times with an organic solvent It was extracted twice. The extract was also washed with saturated brine and then dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was filtered off and the filtrate was concentrated under reduced pressure. Alternatively, the above liquid separation operation may be omitted, and the reaction solution may be concentrated under reduced pressure to proceed to the next silica gel column purification. The obtained residue was purified by silica gel column chromatography [dichloromethane/methanol, ethyl acetate/methanol, etc.] to obtain compound (4b). If necessary, after dissolving the obtained compound (4b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol or a mixed solvent thereof), a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) is added to obtain The purity can be raised by re-precipitation and filtering the solid.

(B-3 공정) (Step B-3)

본 공정은, 식 (4b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 에스테르화를 실시함으로써, 식 (5b) 의 화합물을 제조하는 공정이다. This step is a step for producing the compound of the formula (5b) by esterifying the compound of the formula (4b) using a known organic chemical method.

화합물 (4b) 를, 용매 (N,N-디메틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈, 아세토니트릴 등) 중에서, 5 ℃ 부터 35 ℃ 까지에 있어서, N-하이드록시숙신이미드 및 축합제 (1-에틸-3-(3-디메틸아미노프로필)카르보디이미드염산염 등) 과 반응시킴으로써 에스테르화를 실시하였다. N-하이드록시숙신이미드 및 축합제는, 화합물 (4b) 1 몰에 대하여, 각각 1 몰 내지 3 몰 사용하였다. 반응 시간은 30 분간 내지 72 시간, 바람직하게는 2 시간 내지 24 시간이다. 반응액을 유기 용매 (디클로로메탄, 클로로포름, 아세트산에틸 또는 그 혼합 용매) 로 엷게 한 후, 빙수 (氷水) 로 3 회 내지 5 회 세정하였다. 유기층을 무수 염 (무수 황산나트륨 또는 무수 황산마그네슘) 을 사용하여 건조시켰다. 건조제를 여과 제거한 후, 여과액을 감압하에서 농축하여 화합물 (5b) 의 미정제체를 얻었다. 필요에 따라, 얻어진 화합물 (5b) 를 C18 실리카 겔 칼럼 크로마토그래피 [아세토니트릴만] 으로 정제해도 된다. 또, 얻어진 화합물 (5b) 를 양용매 (아세트산에틸, 아세토니트릴, 디클로로메탄 또는 그들의 혼합 용매) 에 용해한 후, 빈용매 (디에틸에테르, 디이소프로필에테르, 헥산 등) 를 첨가하여 재침전하고, 고체를 여과 채취함으로써 순도를 올릴 수 있다.Compound (4b) is diluted with N-hydroxy in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at 5°C to 35°C. Esterification was carried out by reacting with succinimide and a condensing agent (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). N-hydroxysuccinimide and the condensing agent were used in an amount of 1 to 3 moles, respectively, based on 1 mole of the compound (4b). The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. After thinning the reaction solution with an organic solvent (dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof), it was washed 3 to 5 times with ice water. The organic layer was dried using anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering off the drying agent, the filtrate was concentrated under reduced pressure to obtain a crude compound (5b). If necessary, the obtained compound (5b) may be purified by C18 silica gel column chromatography [acetonitrile only]. Further, after dissolving the obtained compound (5b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane or a mixed solvent thereof), reprecipitation was performed by adding a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) to form a solid Purity can be increased by filtering.

(B-4 공정) (Step B-4)

본 공정은, 식 (5b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여, 식 (6b) 의 화합물과의 축합 반응을 실시함으로써, 식 (2) 의 화합물을 제조하는 공정이다.This step is a step for producing the compound of formula (2) by subjecting the compound of formula (5b) to a condensation reaction with the compound of formula (6b) using a known organic chemical method.

화합물 (6b) 를 용매 중 (N,N-디메틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈, 아세토니트릴 등) 에서, -10 ℃ 부터 100 ℃ 까지, 바람직하게는 15 ℃ 부터 35 ℃ 까지에 있어서, 염기 (트리에틸아민, N,N-디이소프로필에틸아민 등) 및 화합물 (5b) 와 반응시킴으로써 축합 반응을 실시하였다. 화합물 (6b) 1 몰에 대하여, 염기는 2 몰 내지 5 몰 사용하고, 화합물 (5b) 는 1 몰 내지 2 몰 사용하였다. 반응 시간은 5 분간 내지 24 시간, 바람직하게는 1 시간 내지 6 시간이다. 반응액에 벤질아민을 첨가하여 반응 정지 처리를 하였다. 벤질아민은 화합물 (6b) 1 몰에 대하여 4 몰 내지 10 몰 사용하였다. 필요에 따라 반응액을 감압하에서 부분적으로 농축하고, 잔류한 용액을 분취 HPLC [완충액/아세토니트릴, 완충액/메탄올 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴, 완충액/메탄올 등] 또는 그것들을 조합함으로써 정제하고, 화합물 (2) 를 얻었다.Compound (6b) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) from -10°C to 100°C, preferably 15°C to 35°C, a condensation reaction was carried out by reacting with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (5b). Based on 1 mole of compound (6b), 2 to 5 moles of the base were used, and 1 to 2 moles of compound (5b) was used. The reaction time is 5 minutes to 24 hours, preferably 1 hour to 6 hours. Benzylamine was added to the reaction mixture to stop the reaction. 4 to 10 moles of benzylamine were used per mole of compound (6b). If necessary, the reaction solution was partially concentrated under reduced pressure, and the remaining solution was preparative HPLC [buffer/acetonitrile, buffer/methanol, etc.], C18 silica gel column chromatography [buffer/acetonitrile, buffer/methanol, etc.] or those were purified by combining them to obtain compound (2).

B' 법 : 콘쥬게이션 전구체 (시스테인 콘쥬게이션) Method B': conjugation precursor (cysteine conjugation)

B' 법 B' method

본 발명의 (2') 로 나타내는 콘쥬게이션 전구체는, 이하에 기재하는 B' 법에 따라서 제조할 수 있다.The conjugated precursor represented by (2') of the present invention can be produced according to method B' described below.

[화학식 97][Formula 97]

Figure pat00097
Figure pat00097

본 제조법은, L1 의 임의의 위치에 -NH2 가 치환되어 있는 경우의 콘쥬게이션 전구체 (2') 를 제조하기 위한 방법이다.This production method is a method for producing a conjugation precursor (2') in which -NH 2 is substituted at any position of L 1 .

[화학식 98][Formula 98]

Figure pat00098
Figure pat00098

(B-5 공정) (Step B-5)

본 공정은, 식 (2b') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (7b) 의 화합물과의 아미드화를 실시함으로써, 식 (8b) 의 화합물을 제조하는 공정이다. 염기를 사용하지 않는 것 이외는, B 법 B-2 공정에 기재된 수법에 따라서 화합물 (8b) 를 얻었다.This step is a step for producing a compound of formula (8b) by subjecting the compound of formula (2b') to amidation with a compound of formula (7b) using a known organic chemical method. Compound (8b) was obtained according to the method described in Method B, Step B-2, except that no base was used.

(B-6 공정) (Step B-6)

본 공정은, 식 (8b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 보호기를 제거함으로써 식 (9b) 의 화합물을 제조하는 공정이다. PRO6 이 tert-부틸기인 경우, 정제 조작에 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 을 사용하는 것 이외는, B 법 B-1 공정에 기재된 수법에 따라서 화합물 (9b) 를 얻었다This step is a step for preparing the compound of formula (9b) by removing the protecting group from the compound of formula (8b) using a known organic chemical method. When PRO 6 is a tert-butyl group, compound (9b) was obtained according to the method described in Method B, Step B-1, except that silica gel column chromatography [dichloromethane/methanol] was used for purification.

(B-7 공정) (B-7 process)

본 공정은, 식 (9b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 에스테르화를 실시함으로써, 식 (10b) 의 화합물을 제조하는 공정이다. B 법 B-3 공정에 기재된 수법에 따라서 화합물 (10b) 를 얻었다.This step is a step for producing the compound of the formula (10b) by esterifying the compound of the formula (9b) using a known organic chemical method. Method B Compound (10b) was obtained according to the method described in step B-3.

(B-8 공정) (B-8 process)

본 공정은, 식 (6b) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여, 식 (10b) 의 화합물과의 축합 반응을 실시함으로써, 식 (2') 의 화합물을 제조하는 공정이다. B 법 B-4 공정에 기재된 수법에 따라서 화합물 (2') 를 얻었다.This step is a step for producing the compound of formula (2') by subjecting the compound of formula (6b) to a condensation reaction with the compound of formula (10b) using a known organic chemical method. Method B Compound (2') was obtained according to the method described in Step B-4.

C 법 C method

본 발명의 (3) 으로 나타내는 콘쥬게이션 전구체는, 이하에 기재하는 C 법에 따라서 제조할 수 있다.The conjugation precursor represented by (3) of the present invention can be produced according to method C described below.

[화학식 99][Formula 99]

Figure pat00099
Figure pat00099

본 제조법은, L1 의 임의의 위치에 하이드록시기가 치환되어 있는 경우의 콘쥬게이션 전구체 (3) 을 제조하기 위한 방법이다.This production method is a method for producing a conjugation precursor (3) in which a hydroxyl group is substituted at any position of L 1 .

[화학식 100][Formula 100]

Figure pat00100
Figure pat00100

(C-1 공정) (C-1 process)

본 공정은, 식 (1c) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (2c) 의 화합물과의 아미드화를 실시함으로써, 식 (3c) 의 화합물을 제조하는 공정이다. B 법 B-2 공정에 기재된 수법에 따라서 화합물 (3c) 를 얻었다.This step is a step for producing a compound of formula (3c) by subjecting the compound of formula (1c) to amidation with a compound of formula (2c) using a known organic chemical method. Method B Compound (3c) was obtained according to the method described in step B-2.

(C-2 공정) (C-2 process)

본 공정은, 식 (3c) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 에스테르화를 실시함으로써, 식 (4c) 의 화합물을 제조하는 공정이다. B 법 B-3 공정에 기재된 수법에 따라서 화합물 (4c) 를 얻었다.This step is a step for producing the compound of the formula (4c) by esterifying the compound of the formula (3c) using a known organic chemical method. Method B Compound (4c) was obtained according to the method described in step B-3.

(C-3 공정) (C-3 process)

본 공정은, 식 (5c) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (6c) 의 화합물과의 커플링 반응 (아미노메틸렌화) 과 얻어진 커플링체의 탈보호를 연속해서 실시함으로써, 식 (7c) 의 화합물을 제조하는 공정이다.In this step, the compound of the formula (5c) is continuously subjected to a coupling reaction (aminomethyleneization) with the compound of the formula (6c) and deprotection of the obtained coupling body using a known organic chemical method to obtain the formula This is a step for producing the compound of (7c).

PRO9 가 9-플루오레닐메틸옥시카르보닐기인 경우, 화합물 (5c) 를, 테트라하이드로푸란 중에서, 5 ℃ 부터 35 ℃ 까지에 있어서, 화합물 (6c) 및 산 (p-톨루엔술폰산 등) 과 반응시킴으로써 아미노메틸렌화를 실시하였다. 화합물 (5c) 1 몰에 대하여, 화합물 (6c) 는 1 몰 내지 20 몰, 바람직하게는 2 몰 내지 10 몰 사용하고, 산은 0.05 몰 내지 과잉 몰, 바람직하게는 0.1 몰 내지 3 몰 사용하였다. 반응 시간은 30 분간 내지 72 시간, 바람직하게는 2 시간 내지 24 시간이다. 이어서, 반응액에 염기 (1,8-디아자비시클로[5.4.0]-7-운데센 등) 를 첨가하고, 탈보호를 실시하였다. 반응액이 현탁해 있는 경우에는, 필요에 따라 용매 (N,N-디메틸포름아미드 등) 를 추가하여 용해시킨 후에 반응시킬 수 있다. 염기는 화합물 (5c) 1 몰에 대하여 과잉 몰, 바람직하게는 5 몰 내지 20 몰 사용하였다. 반응 시간은 10 분간 내지 24 시간, 바람직하게는 2 시간 내지 12 시간이다. 반응액에 물을 첨가하여 그대로 C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴 등] 으로 정제하고, 화합물 (7c) 를 얻었다.When PRO 9 is a 9-fluorenylmethyloxycarbonyl group, compound (5c) is reacted with compound (6c) and an acid (such as p-toluenesulfonic acid) in tetrahydrofuran at 5°C to 35°C. Aminomethyleneation was performed. For 1 mole of compound (5c), 1 to 20 moles, preferably 2 to 10 moles of compound (6c) were used, and 0.05 mole to excess mole, preferably 0.1 to 3 moles of acid was used. The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. Then, a base (such as 1,8-diazabicyclo[5.4.0]-7-undecene) was added to the reaction mixture to deprotect. When the reaction solution is suspended, it can be reacted after dissolving by adding a solvent (such as N,N-dimethylformamide) as necessary. The base was used in an excess mole, preferably 5 to 20 moles, based on 1 mole of compound (5c). The reaction time is 10 minutes to 24 hours, preferably 2 hours to 12 hours. Water was added to the reaction mixture and purified as it was by C18 silica gel column chromatography [buffer/acetonitrile, etc.] to obtain compound (7c).

(C-4 공정) (C-4 process)

본 공정은, 식 (7c) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 보호기를 제거하고, 식 (8c) 의 화합물을 제조하는 공정이다. PRO7 및 PRO8 이 tert-부틸디메틸실릴기인 경우, A 법 A-7 공정에 기재된 수법에 따라서 화합물 (8c) 를 얻었다.This step is a step of preparing a compound of formula (8c) by removing a protecting group from the compound of formula (7c) using a known organic chemical method. When PRO 7 and PRO 8 are tert-butyldimethylsilyl groups, compound (8c) was obtained according to the method described in method A, step A-7.

(C-5 공정) (C-5 process)

본 공정은, 식 (8c) 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여, 식 (4c) 의 화합물과의 축합 반응을 실시함으로써, 식 (3) 의 화합물을 제조하는 공정이다. B 법 B-4 공정에 기재된 수법에 따라서 화합물 (3) 을 얻었다.This step is a step for producing the compound of the formula (3) by subjecting the compound of the formula (8c) to a condensation reaction with the compound of the formula (4c) using a known organic chemical method. Method B Compound (3) was obtained according to the method described in Step B-4.

C' 법 C' method

본 발명의 (3') 로 나타내는 콘쥬게이션 전구체는, 이하에 기재하는 C' 법에 따라서 제조할 수 있다.The conjugation precursor represented by (3') of the present invention can be produced according to method C' described below.

[화학식 101][Formula 101]

Figure pat00101
Figure pat00101

본 제조법은, L1 의 임의의 위치에 하이드록시기가 치환되어 있는 경우의 콘쥬게이션 전구체 (3') 를 제조하기 위한 방법이다.This production method is a method for producing a conjugation precursor (3') in which a hydroxyl group is substituted at any position of L 1 .

[화학식 102][Formula 102]

Figure pat00102
Figure pat00102

(C'-1 공정) (C'-1 process)

본 공정은, 식 (1c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 가수분해 반응과 시아노에틸기의 제거를 연속해서 실시함으로써, 식 (2c') 의 화합물을 제조하는 공정이다. A 법 A-1 공정에 기재된 수법에 따라서 화합물 (2c') 를 얻었다.This step is a step for producing the compound of the formula (2c') by successively subjecting the compound of the formula (1c') to a hydrolysis reaction and removal of a cyanoethyl group using a known organic chemical method. Method A Compound (2c') was obtained according to the method described in Step A-1.

(C'-2 공정) (C'-2 process)

본 공정은, 식 (2c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 하이드록시기의 보호기를 제거하고, 식 (3c') 의 화합물을 제조하는 공정이다. A 법 A-2 공정에 기재된 수법에 따라서 화합물 (3c') 를 얻었다.This step is a step of preparing the compound of formula (3c') by removing the protecting group of the hydroxyl group from the compound of formula (2c') using a known organic chemical method. Method A Compound (3c') was obtained according to the method described in Step A-2.

(C'-3 공정) (C'-3 process)

본 공정은, 식 (3c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 식 (4c') 의 화합물과의 커플링 반응 및 얻어진 커플링체의 황화 반응 또는 산화 반응을 연속해서 실시함으로써, 식 (5c') 의 화합물을 제조하는 공정이다. A 법 A-3 공정 또는 A'' 법 A''-3 공정에 기재된 수법에 따라서 화합물 (5c') 를 얻었다.In this step, the compound of formula (3c') is continuously subjected to a coupling reaction with the compound of formula (4c') and a sulfurization reaction or oxidation reaction of the obtained coupling body using a known organic chemical method, This is a step for producing the compound of (5c'). Compound (5c') was obtained according to the method described in method A step A-3 or method A″ step A″-3.

(C'-4 공정) (C'-4 process)

본 공정은, 식 (5c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 하이드록시기의 보호기를 제거하고, 식 (6c') 의 화합물을 제조하는 공정이다. A 법 A-4 공정에 기재된 수법에 따라서 화합물 (6c') 를 얻었다.This step is a step of preparing the compound of formula (6c') by removing the protecting group of the hydroxyl group from the compound of formula (5c') using a known organic chemical method. Method A Compound (6c') was obtained according to the method described in step A-4.

(C'-5 공정) (C'-5 process)

본 공정은, 식 (6c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 고리화 반응과 황화 반응 또는 산화 반응을 연속해서 실시하고, 식 (7c') 의 화합물을 제조하는 공정이다. A 법 A-5 공정 또는 A' 법 A'-5 공정에 기재된 수법에 따라서 화합물 (7c') 를 얻었다.This step is a step of producing a compound of formula (7c') by successively subjecting the compound of formula (6c') to a cyclization reaction and sulfurization or oxidation reaction using a known organic chemical method. Compound (7c') was obtained according to the method described in Method A Step A-5 or Method A' Step A'-5.

(C'-6 공정) (C'-6 process)

본 공정은, 식 (7c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 시아노에틸기 및 모든 아실계 보호기를 동시에 제거하고, 식 (8c') 의 화합물을 제조하는 공정이다. A 법 A-6 공정에 기재된 수법에 따라서 화합물 (8c') 를 얻었다.This step is a step for preparing the compound of formula (8c') by simultaneously removing the cyanoethyl group and all acyl protecting groups from the compound of formula (7c') using a known organic chemical method. Method A Compound (8c') was obtained according to the method described in step A-6.

(C'-7 공정) (C'-7 process)

본 공정은, 식 (8c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여 모든 실릴계 보호기를 동시에 제거하고, 식 (9c') 의 화합물을 제조하는 공정이다. PRO9 가 2-(트리메틸실릴)에톡시카르보닐기인 경우, 화합물 (8c') 를, 5 ℃ 부터 100 ℃ 까지, 바람직하게는 35 ℃ 부터 60 ℃ 까지에 있어서, 불화테트라부틸암모늄의 테트라하이드로푸란 용액으로 처리함으로써, 2-(트리메틸실릴)에톡시카르보닐기를 제거하였다. 불화테트라부틸암모늄은, 화합물 (8c') 1 몰에 대하여 과잉 몰, 바람직하게는 10 내지 30 몰 사용하였다. 반응 시간은 1 시간 내지 48 시간, 바람직하게는 4 시간 내지 24 시간이다. 반응액에 완충액을 첨가하여 희석 후, 필요에 따라 유기 용매 성분을 감압하에서 증류 제거하였다. 잔류물을 분취 HPLC [완충액/아세토니트릴, 완충액/메탄올 등], C18 실리카 겔 칼럼 크로마토그래피 [완충액/아세토니트릴, 완충액/메탄올 등] 또는 그것들을 조합함으로써 정제하고, 화합물 (9c') 를 얻었다.This step is a step for preparing a compound of formula (9c') by simultaneously removing all silyl protecting groups from the compound of formula (8c') using a known organic chemical method. When PRO 9 is a 2-(trimethylsilyl)ethoxycarbonyl group, compound (8c') is dissolved in a tetrahydrofuran solution of tetrabutylammonium fluoride at 5°C to 100°C, preferably at 35°C to 60°C. By treatment with , the 2-(trimethylsilyl)ethoxycarbonyl group was removed. Tetrabutylammonium fluoride was used in an excess mole, preferably 10 to 30 moles, per mole of compound (8c'). The reaction time is 1 hour to 48 hours, preferably 4 hours to 24 hours. After dilution by adding a buffer to the reaction solution, organic solvent components were distilled off under reduced pressure if necessary. The residue was purified by preparative HPLC [buffer/acetonitrile, buffer/methanol, etc.], C18 silica gel column chromatography [buffer/acetonitrile, buffer/methanol, etc.] or a combination thereof to obtain compound (9c').

(C'-8 공정) (C'-8 process)

본 공정은, 식 (9c') 의 화합물에 대하여 공지된 유기 화학적 수법을 이용하여, 식 (4c) 의 화합물과의 축합 반응을 실시함으로써, 식 (3') 의 화합물을 제조하는 공정이다. B 법 B-4 공정에 기재된 수법에 따라서 화합물 (3') 를 얻었다.This step is a step for producing the compound of formula (3') by subjecting the compound of formula (9c') to a condensation reaction with the compound of formula (4c) using a known organic chemical method. Method B Compound (3') was obtained according to the method described in Step B-4.

D 법 : 당사슬 리모델링 항체의 제조 Method D: Preparation of oligosaccharide remodeling antibody

당사슬 리모델링 항체는, 예를 들어 WO2018/003983 등에 기재된 방법에 준하여, 다음 식에 나타내는 방법으로 제조할 수 있다.A sugar chain remodeling antibody can be produced by the method shown in the following formula, for example, according to the method described in WO2018/003983 and the like.

[화학식 103][Formula 103]

Figure pat00103
Figure pat00103

(D-1 공정) (D-1 process)

본 공정은, 목적으로 하는 항체에 대하여, 공지된 효소 반응을 이용하여 항체의 아미노산 서열 297 번째의 아스파라긴에 결합하는 N 결합형 당사슬 (N297 결합 당사슬) 의 환원 말단 키토비오스 구조의 GlcNAcβ1 - 4GlcNAc 사이의 글리코시드 결합을 가수분해에 의해 절단하고, 당사슬 절단 항체를 제조하는 공정이다. 목적으로 하는 항체 (1d) (10 ㎎/㎖) 를 완충액 (인산 완충액 등) 중, 0 ℃ 부터 40 ℃ 까지에 있어서, 야생형 EndoS 효소 등의 가수분해 효소를 사용하여 환원 말단의 키토비오스 구조의 GlcNAcβ1 과 4GlcNAc 사이의 글리코시드 결합의 가수분해 반응을 실시하였다. 반응 시간은 10 분 내지 72 시간, 바람직하게는 1 시간 내지 6 시간이다. 야생형 EndoS 효소는 항체 (1d) 100 ㎎ 에 대하여, 0.1 ㎎ 내지 10 ㎎, 바람직하게는 0.1 ㎎ 내지 3 ㎎ 을 사용하였다. 반응 종료 후, 어피니티 크로마토그래피 (HiTrap rProtein A FF (5 ㎖) (GE 헬스 케어 제조)) 및/또는 하이드록시 아파타이트 칼럼 (Bio-Scale Mini CHT Type I 카트리지 (5 ㎖) (BIO-RAD 제조)) 으로 정제하고, (Fucα1,6)GlcNAc 항체 (2d) 를 얻었다.In this step, for the antibody of interest, using a known enzymatic reaction, the N-linked sugar chain (N297-linked sugar chain) that binds to asparagine at position 297 of the antibody amino acid sequence between GlcNAcβ1 and 4GlcNAc of the chitobiose structure at the reducing end This is a process in which glycosidic bonds are cleaved by hydrolysis to produce glycoside-cleaved antibodies. Antibody of interest (1d) (10 mg/ml) was incubated in a buffer solution (phosphate buffer, etc.) at 0°C to 40°C using a hydrolase such as wild-type EndoS enzyme to reduce GlcNAcβ1 of chitobiose structure at the reduced end. A hydrolysis reaction of the glycosidic bond between and 4GlcNAc was performed. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, based on 100 mg of antibody (1d). After completion of the reaction, affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE Healthcare)) and / or hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (manufactured by BIO-RAD) ) to obtain a (Fucα1,6)GlcNAc antibody (2d).

(D-2 공정) (D-2 process)

본 공정은, D-1 공정에서 얻어진 (Fucα1,6)GlcNAc 항체 (2d) 에 대하여, 공지된 효소 반응을 이용하여 아지드기를 포함하는 PEG 링커를 갖는 SG 형 또는 MSG (MSG1, MSG2) 형 당사슬 옥사졸린체 (이하, 「아지드 당사슬 옥사졸린체」) 를 결합시켜, 당사슬 리모델링 항체 (3d) 를 제조하는 공정이다.In this step, with respect to the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1, using a known enzyme reaction, SG-type or MSG (MSG1, MSG2)-type sugar chains having an azide group-containing PEG linker are prepared. This is a step for producing a sugar chain remodeling antibody (3d) by binding an oxazoline body (hereinafter referred to as "azide sugar chain oxazoline body").

항체 (2d) 를 완충액 (인산 완충액 등) 중, 0 ℃ 부터 40 ℃ 까지에 있어서, EndoS (D233Q/Q303L) 등의 당전이 효소 존재하, 아지드 당사슬 옥사졸린체와 반응시킴으로써, 당사슬 전이 반응을 실시하였다. 반응 시간은 10 분 내지 72 시간, 바람직하게는 1 시간 내지 6 시간이다. EndoS 효소 (D233Q/Q303L) 는 항체 100 ㎎ 에 대하여, 1 ㎎ 내지 10 ㎎, 바람직하게는 1 ㎎ 내지 3 ㎎ 을 사용하고, 아지드 당사슬 옥사졸린체는 2 당량 내지 과잉 당량, 바람직하게는 4 당량 내지 20 당량 사용하였다. 반응 종료 후, 어피니티 크로마토그래피 (HiTrap rProtein A FF (5 ㎖) (GE 헬스 케어 제조)) 및 하이드록시 아파타이트 칼럼 (Bio-Scale Mini CHT Type I 카트리지 (5 ㎖) (BIO-RAD 제조)) 으로 정제하고, 당사슬 리모델링 항체 (3d) 를 얻었다.By reacting the antibody (2d) with an azide oligosaccharide oxazoline in a buffer (phosphate buffer or the like) at 0°C to 40°C in the presence of a glycosyltransferase such as EndoS (D233Q/Q303L), the glycosyltransfer reaction is conducted. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. The EndoS enzyme (D233Q/Q303L) is used in an amount of 1 mg to 10 mg, preferably 1 mg to 3 mg, based on 100 mg of the antibody, and the azide oligosaccharide oxazoline body is 2 equivalents to excess equivalents, preferably 4 equivalents. to 20 equivalents were used. After completion of the reaction, affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE Healthcare)) and hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (manufactured by BIO-RAD)) After purification, a sugar chain remodeling antibody (3d) was obtained.

상기의 당사슬 리모델링 항체의 조제에 있어서, 항체 수용액의 농축, 농도 측정, 그리고 버퍼 교환은, 후술하는 공통 조작 A 내지 C 에 따라서 실시할 수 있다. In the preparation of the oligosaccharide remodeling antibody described above, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to common operations A to C described later.

또한, SG 형의 아지드 당사슬 옥사졸린체는 WO2018/003983 에 기재된 방법에 준하여 합성하였다. 일례로서 [N3-PEG(3)]2-SG(10)-Ox (WO2018/003983 에 기재된 화합물 1-10) 의 합성 방법을 다음 식에 나타낸다.In addition, the azide oligosaccharide oxazoline body of the SG type was synthesized according to the method described in WO2018/003983. As an example, the synthesis method of [N 3 -PEG(3)] 2 -SG(10)-Ox (Compound 1-10 described in WO2018/003983) is shown in the following formula.

[화학식 104][Formula 104]

Figure pat00104
Figure pat00104

MSG 형의 아지드 당사슬 옥사졸린체도 WO2018/003983 에 기재된 방법에 준하여 합성하였다. 일례로서 [N3-PEG(3)]-MSG1(9)-Ox (WO2018/003983 에 기재된 화합물 1-11) 의 합성 방법을 다음 식에 나타낸다.A MSG-type azide oligosaccharide oxazoline was also synthesized according to the method described in WO2018/003983. As an example, the synthesis method of [N 3 -PEG(3)]-MSG1(9)-Ox (Compound 1-11 described in WO2018/003983) is shown in the following formula.

[화학식 105][Formula 105]

Figure pat00105
Figure pat00105

E 법 : 항체와 약물의 콘쥬게이션 (당사슬 콘쥬게이션 1) Method E: conjugation of antibody and drug (sugar chain conjugation 1)

[화학식 106][Formula 106]

Figure pat00106
Figure pat00106

(여기서, 항체 약물 콘쥬게이트 (1e) 의 좌측의 2 개의 아스테리스크 (*) 는 우측의 아스테리스크로 나타내는 약물 링커 부분을 나타낸다.) (Here, the two asterisks (*) on the left side of the antibody drug conjugate (1e) represent the drug linker moiety indicated by the right asterisk.)

본 제조법은, D 법 D-2 공정에서 얻어진 당사슬 리모델링 항체 (3d) 와 B 법 B-4 공정에서 얻어진 콘쥬게이션 전구체 (2) 를 SPAAC (strain-promoted azide-alkyne cycloaddition : J. Am. Chem. Soc. 2004, 126, 15046-15047) 반응에 의해 결합시켜, 항체 약물 콘쥬게이트 (1e) 를 제조하는 방법이다.In this production method, the sugar chain remodeling antibody (3d) obtained in step D-2 of method D and the conjugated precursor (2) obtained in step B-4 of method B were mixed with SPAAC (strain-promoted azide-alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046-15047) reaction to prepare antibody drug conjugate (1e).

(E-1 공정) (E-1 process)

당사슬 리모델링 항체 (3d) 의 완충 용액 (인산 완충액, 아세트산 완충액, 붕산 완충액 등) 과, 콘쥬게이션 전구체 (2) 를 적당한 용매 (디메틸술폭시드, N,N-디메틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈, 프로필렌글리콜 또는 그들의 혼합 용매) 에 용해시킨 용액을 혼합함으로써, SPAAC 반응을 실시하였다. 콘쥬게이션 전구체 (2) 는, 당사슬 리모델링 항체 (3d) 1 몰에 대하여, 2 몰 내지 과잉 몰, 바람직하게는 4 몰 내지 30 몰이며, 유기 용매의 비율은, 항체의 완충 용액에 대하여 1 ％ 내지 200 ％ (v/v) 가 바람직하다. 반응 온도는 0 ℃ 내지 37 ℃, 바람직하게는 15 ℃ 내지 25 ℃ 이며, 반응 시간은 1 시간 내지 150 시간, 바람직하게는 6 시간 내지 72 시간이다. 반응 용액의 pH 는 5 내지 9 가 바람직하다. 반응 용액을 후술하는 공통 조작 D 에 기재된 방법에 따라서 정제하고, 항체 약물 콘쥬게이트 (1e) 를 얻었다.A buffer solution (phosphate buffer, acetate buffer, boric acid buffer, etc.) of the sugar chain remodeling antibody (3d) and the conjugation precursor (2) are mixed with an appropriate solvent (dimethylsulfoxide, N,N-dimethylformamide, N,N-dimethylacetate) SPAAC reaction was performed by mixing a solution dissolved in amide, N-methylpyrrolidone, propylene glycol or a mixed solvent thereof). Conjugation precursor (2) is 2 mol to excess mol, preferably 4 mol to 30 mol, based on 1 mol of sugar chain remodeling antibody (3d), and the ratio of the organic solvent is 1% to 1% with respect to the antibody buffer solution. 200% (v/v) is preferred. The reaction temperature is 0°C to 37°C, preferably 15°C to 25°C, and the reaction time is 1 hour to 150 hours, preferably 6 hours to 72 hours. As for pH of a reaction solution, 5-9 are preferable. The reaction solution was purified according to the method described in common operation D described later to obtain antibody drug conjugate (1e).

E' 법 : 항체와 약물의 콘쥬게이션 (시스테인 콘쥬게이션) E' method: conjugation of antibody and drug (cysteine conjugation)

시스테인 콘쥬게이션을 갖는 본 발명의 항체 약물 콘쥬게이트는, 참고예 1 등에 따라서 조제한 목적으로 하는 항체와 B' 법 B-8 공정에서 얻어진 말레이미드기를 갖는 콘쥬게이션 전구체 (2') 를 사용하여, WO2014/057687 등에 기재된 방법에 준하여 제조할 수 있다.The antibody drug conjugate of the present invention having cysteine conjugation is WO2014, using the target antibody prepared according to Reference Example 1 and the like and the conjugation precursor (2') having a maleimide group obtained in step B-8 of method B'. It can be manufactured according to the method described in /057687 et al.

E'' 법 : 항체와 약물의 콘쥬게이션 (당사슬 콘쥬게이션 2) E'' method: conjugation of antibody and drug (sugar chain conjugation 2)

E 법에 있어서, 콘쥬게이션 전구체 (2) 를 C' 법 C'-8 공정에서 얻어진 콘쥬게이션 전구체 (3') 로 바꿈으로써, 다음 식에 나타내는 항체 약물 콘쥬게이트 (1e'') 를 얻었다.In method E, the antibody drug conjugate (1e″) shown in the following formula was obtained by replacing the conjugation precursor (2) with the conjugation precursor (3′) obtained in method C′, step C′-8.

[화학식 107][Formula 107]

Figure pat00107
Figure pat00107

(여기서, 항체 약물 콘쥬게이트 (1e'') 의 좌측의 2 개의 아스테리스크 (*1) 는 우측의 아스테리스크로 나타내는 약물 링커 부분을 나타낸다.) (Here, the two asterisks (*1) on the left side of the antibody drug conjugate (1e'') represent the drug linker moiety indicated by the right asterisk.)

항체 약물 콘쥬게이트는, 후술하는 공통 조작 D 내지 G 에 의해 버퍼 교환, 정제, 항체 농도의 측정 및 항체 1 분자당의 약물 평균 결합수의 측정을 실시하고, 항체 약물 콘쥬게이트의 동정 (同定) 을 실시할 수 있다.For the antibody drug conjugate, the antibody drug conjugate is identified by carrying out buffer exchange, purification, measurement of the antibody concentration, and measurement of the average number of drug bonds per antibody molecule by common operations D to G described later. can do.

공통 조작 A : 항체 수용액의 농축 Common Operation A: Concentration of Antibody Aqueous Solution

Amicon (등록상표) Ultra 원심식 필터 디바이스 (50,000 NMWL, Merck Millipore Ltd.) 에 항체 또는 항체 약물 콘쥬게이트 용액을 넣고, 원심기 (Allegra X-15R, Beckman Coulter, Inc.) 를 사용한 원심 조작 (2000 G 내지 4000 G 로 5 분간 내지 20 분간 원심) 에 의해, 항체 및 항체 약물 콘쥬게이트 용액을 농축하였다.An antibody or antibody drug conjugate solution was put into an Amicon (registered trademark) Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and centrifugal manipulation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000 Centrifugation at G to 4000 G for 5 minutes to 20 minutes) to concentrate the antibody and antibody drug conjugate solutions.

공통 조작 B : 항체의 농도 측정 Common Operation B: Determination of Antibody Concentration

UV 측정기 (Nanodrop 1000, Thermo Fisher Scientific, Inc.) 를 사용하여, 메이커 규정의 방법에 따라서, 항체 농도의 측정을 실시하였다. 그 때에, 항체마다 상이한 280 ㎚ 흡광 계수 (1.3 ㎖㎎-1㎝-1 내지 1.8 ㎖㎎-1㎝-1) 를 사용하였다.Antibody concentration was measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's method. At that time, different 280 nm extinction coefficients (1.3 ml mg -1 cm -1 to 1.8 ml mg -1 cm -1 ) for each antibody were used.

공통 조작 C : 항체의 버퍼 교환 Common Operation C: Buffer Exchange of Antibodies

항체 수용액에 완충액 (인산 완충 생리 식염수 (pH 6.0), 인산 완충액 (pH 6.0) 등) 을 첨가하고, 공통 조작 A 에 기재된 방법에 따라서 농축하였다. 이 조작을 수 회 실시한 후, 공통 조작 B 에 기재된 방법에 따라서 항체 농도를 측정하였다. 이 항체 완충 용액에, 적절히 완충액 (인산 완충 생리 식염수 (pH 6.0), 인산 완충액 (pH 6.0) 등) 을 첨가하여, 목적으로 하는 농도 (예를 들어, 약 10 ㎎/㎖) 의 항체 완충 용액을 조제하였다.A buffer (phosphate buffered physiological saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the antibody aqueous solution, and concentrated according to the method described in Common Operation A. After performing this operation several times, the antibody concentration was measured according to the method described in common operation B. To this antibody buffer solution, a buffer (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) is appropriately added to obtain an antibody buffer solution having a target concentration (eg, about 10 mg/ml). prepared.

공통 조작 D : 항체 약물 콘쥬게이트의 정제 (겔 여과 크로마토그래피) Common Operation D: Purification of Antibody Drug Conjugates (Gel Filtration Chromatography)

아세트산 완충액 (10 mM Acetate Buffer, 5 ％ Sorbitol, pH 5.5 ; 본 명세서에서는 ABS 라고 칭한다) 또는 그 이외의 적당한 완충액으로 NAP 칼럼 (NAP-5, NAP-10, NAP-25 (GE 헬스 케어 제조)) 을 평형화시켰다. 이 NAP 칼럼에, 항체 약물 콘쥬게이트 반응 용액을 차지하고, 메이커 규정량의 완충액을 자연 유하시켜, 항체 획분을 분취하였다. 이 획분을 재차 NAP 칼럼에 차지하고, 메이커 규정량의 완충액을 자연 유하시켜, 항체 획분을 분취하였다. 이 조작을 합계 2 회 내지 3 회 반복함으로써, 미결합의 약물 링커나 디메틸술폭시드, 프로필렌글리콜을 제외한 항체 약물 콘쥬게이트를 얻었다. 필요에 따라, 공통 조작 A 및 C 에 의해 항체 약물 콘쥬게이트 용액의 농도를 조절하였다.Acetate buffer (10 mM Acetate Buffer, 5% Sorbitol, pH 5.5; referred to herein as ABS) or other suitable buffers are used for NAP columns (NAP-5, NAP-10, NAP-25 (manufactured by GE Healthcare)) was equilibrated. The antibody drug conjugate reaction solution was charged onto this NAP column, and the buffer solution in the amount prescribed by the manufacturer was allowed to flow naturally, and the antibody fraction was fractionated. This fraction was again loaded onto the NAP column, and the antibody fraction was fractionated by naturally flowing the buffer in the amount specified by the manufacturer. By repeating this operation 2 to 3 times in total, an antibody drug conjugate excluding unbonded drug linkers, dimethyl sulfoxide and propylene glycol was obtained. As needed, the concentration of the antibody drug conjugate solution was adjusted by common operations A and C.

공통 조작 E : 항체 약물 콘쥬게이트에 있어서의 항체 농도 및 항체 1 분자당의 약물 평균 결합수의 측정 (UV 법) Common operation E: Measurement of antibody concentration in antibody drug conjugate and average number of drug bonds per antibody molecule (UV method)

항체 약물 콘쥬게이트에 있어서의 결합 약물 농도는, 흡광 광도계 (UV/VIS Spectrometer Lambda 25, PerkinElmer, Inc.) 를 사용하여, 항체 약물 콘쥬게이트 수용액의 280 ㎚ 및 260 ㎚ (260 ㎚ 이외의 파장을 사용하는 경우도 있다) 의 2 파장에 있어서의 흡광도를 측정한 후에, 하기의 계산을 실시함으로써 산출할 수 있다. 어느 파장에 있어서의 전체 흡광도는 계내에 존재하는 모든 흡수 화학종의 흡광도의 합에 동일 (흡광도의 가성성) 하기 때문에, 항체와 약물의 콘쥬게이션 전후에 있어서, 항체 및 약물의 몰 흡광 계수에 변화가 없다고 가정하면, 항체 약물 콘쥬게이트에 있어서의 항체 농도 및 약물 농도는, 하기의 관계식으로 나타낸다.The concentration of the bound drug in the antibody drug conjugate was determined using an absorption photometer (UV/VIS Spectrometer Lambda 25, PerkinElmer, Inc.) at 280 nm and 260 nm (using wavelengths other than 260 nm) of the antibody drug conjugate aqueous solution. It can be calculated by performing the following calculation after measuring the absorbance at two wavelengths of (in some cases). Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing species present in the system (causticity of absorbance), the molar extinction coefficient of the antibody and drug changes before and after conjugation of the antibody and the drug Assuming that there is no , the antibody concentration and drug concentration in the antibody drug conjugate are represented by the following relational expression.

A280 = AD,280 ＋ AA,280 = εD,280CD ＋εA,280CA 식 (I) A 280 = A D , 280 + A A , 280 = ε D , 280 C D + ε A , 280 C A Formula (I)

A260 = AD,260 ＋ AA,260 = εD,260CD ＋εA,260CA 식 (II) A 260 = A D , 260 + A A , 260 = ε D , 260 C D + ε A , 260 C A Equation (II)

여기서, A280 은 280 ㎚ 에 있어서의 항체 약물 콘쥬게이트 수용액의 흡광도를 나타내고, A260 은 260 ㎚ 에 있어서의 항체 약물 콘쥬게이트 수용액의 흡광도를 나타내고, AA,280 은 280 ㎚ 에 있어서의 항체의 흡광도를 나타내고, AA,260 은 260 ㎚ 에 있어서의 항체의 흡광도를 나타내고, AD,280 은 280 ㎚ 에 있어서의 콘쥬게이트 전구체의 흡광도를 나타내고, AD,260 은 260 ㎚ 에 있어서의 콘쥬게이트 전구체의 흡광도를 나타내고, εA,280 은 280 ㎚ 에 있어서의 항체의 몰 흡광 계수를 나타내고, εA,260 은 260 ㎚ 에 있어서의 항체의 몰 흡광 계수를 나타내고, εD,280 은 280 ㎚ 에 있어서의 콘쥬게이트 전구체의 몰 흡광 계수를 나타내고, εD,260 은 260 ㎚ 에 있어서의 콘쥬게이트 전구체의 몰 흡광 계수를 나타내고, CA 는 항체 약물 콘쥬게이트에 있어서의 항체 농도를 나타내고, CD 는 항체 약물 콘쥬게이트에 있어서의 약물 농도를 나타낸다. 여기서, εA,280, εA,260, εD,280, εD,260 은, 사전에 준비한 값 (계산 추정값 또는 실측값) 이 사용된다. 예를 들어, εA,280 은, 항체의 아미노산 서열로부터, 이미 알려진 계산 방법 (Protein Science, 1995, vol. 4, 2411-2423) 에 의해 추정할 수 있다. εA,260 은, 항체의 UV 측정으로부터 얻어진 실측값과 εA,280 의 추정값으로부터 계산한 값을 사용하였다. 실시예에 있어서, 개변 항HER2 항체의 몰 흡광 계수는, εA,280 = 215380 및 εA,260 = 110117 을 사용하였다. 개변 항LPS 항체의 몰 흡광 계수는, εA,280 = 227300 및 εA,260 = 110710 을 사용하였다. εD,280 및 εD,260 은, 사용하는 콘쥬게이트 전구체를 어느 몰 농도로 용해시킨 용액의 흡광도를 측정함으로써, 랩버트·비어의 법칙 (흡광도 = 몰 농도 × 몰 흡광 계수 × 셀 광로 길이) 에 의해, 얻을 수 있다. 실시예에 있어서의 콘쥬게이트 전구체의 몰 흡광 계수는, 그때마다 UV 측정으로 얻었다. 항체 약물 콘쥬게이트 수용액의 A280 및 A260 을 측정하고, 이들 값을 식 (I) 및 (II) 에 대입하여 연립 방정식을 풀음으로써, CA 및 CD 를 구할 수 있다. 또한 CD 를 CA 로 나눔으로써, 항체 1 분자당의 약물 평균 결합수를 구할 수 있다.Here, A 280 represents the absorbance of the antibody drug conjugate aqueous solution at 280 nm, A 260 represents the absorbance of the antibody drug conjugate aqueous solution at 260 nm, and A A , 280 represents the absorbance of the antibody drug conjugate solution at 280 nm Absorbance is represented, A A , 260 represents the absorbance of the antibody at 260 nm, A D , 280 represents the absorbance of the conjugate precursor at 280 nm, and AD , 260 represents the absorbance of the conjugate at 260 nm. Represents the absorbance of the precursor, ε A , 280 represents the molar extinction coefficient of the antibody at 280 nm, ε A , 260 represents the molar extinction coefficient of the antibody at 260 nm, ε D , 280 represents the molar extinction coefficient of the antibody at 280 nm represents the molar extinction coefficient of the conjugate precursor, ε D , 260 represents the molar extinction coefficient of the conjugate precursor at 260 nm, CA represents the antibody concentration in the antibody drug conjugate, and CD represents The drug concentration in the antibody drug conjugate is shown. Here, for ε A , 280 , ε A , 260 , ε D , 280 , ε D , 260 , values prepared in advance (calculated estimated values or measured values) are used. For example, ε A , 280 can be estimated from the amino acid sequence of an antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). For ε A , 260 , a value calculated from the measured value obtained from UV measurement of the antibody and the estimated value of ε A , 280 was used. In the examples, ε A , 280 = 215380 and ε A , 260 = 110117 were used as the molar extinction coefficients of the modified anti-HER2 antibody. As the molar extinction coefficients of the modified anti-LPS antibody, ε A , 280 = 227300 and ε A , 260 = 110710 were used. ε D , 280 and ε D , 260 are obtained by measuring the absorbance of a solution in which the conjugate precursor used is dissolved at a certain molar concentration, and Labbert-Beer's law (absorbance = molar concentration × molar extinction coefficient × cell optical path length) can be obtained by Molar extinction coefficients of the conjugate precursors in Examples were obtained by UV measurement each time. CA and CD can be obtained by measuring A 280 and A 260 of the antibody drug conjugate aqueous solution and substituting these values into equations (I) and (II) to solve simultaneous equations . Also, by dividing C D by C A , the average number of drug bonds per antibody molecule can be obtained.

공통 조작 F : 항체 약물 콘쥬게이트에 있어서의 항체 농도 및 항체 1 분자당의 약물 평균 결합수의 측정 (역상 고속 액체 크로마토그래피법 : RP-HPLC) Common operation F: Measurement of antibody concentration and average number of drug bonds per antibody molecule in antibody drug conjugate (reverse phase high performance liquid chromatography method: RP-HPLC)

항체 약물 콘쥬게이트에 있어서의 항체 농도 및 항체 1 분자당의 약물 평균 결합수는, 전술한 공통 조작 E 에 더하여, 이하의 방법을 사용하는 고속 액체 크로마토그래피 분석에 의해 구할 수 있다.The antibody concentration and the average number of drug bonds per antibody molecule in the antibody drug conjugate can be obtained by high-performance liquid chromatography analysis using the following method in addition to common operation E described above.

[F-1. HPLC 분석용 샘플의 조제 (항체 약물 콘쥬게이트의 환원)] [F-1. Preparation of sample for HPLC analysis (reduction of antibody drug conjugate)]

항체 약물 콘쥬게이트 용액 (약 1 ㎎/㎖, 60 ㎕) 을 디티오트레이톨 (DTT) 수용액 (100 mM, 15 ㎕) 과 혼합하였다. 혼합물을 37 ℃ 에서 30 분간 인큐베이트하여, 항체 약물 콘쥬게이트의 L 사슬 및 H 사슬 사이의 디술파이드 결합을 절단하였다. 이 반응 용액을 그대로 HPLC 분석에 사용하였다.The antibody drug conjugate solution (about 1 mg/ml, 60 μl) was mixed with dithiothreitol (DTT) aqueous solution (100 mM, 15 μl). The mixture was incubated at 37°C for 30 minutes to cleave the disulfide bond between the L chain and the H chain of the antibody drug conjugate. This reaction solution was used for HPLC analysis as it was.

[F-2. HPLC 분석] [F-2. HPLC analysis]

대표적인 분석 조건은 하기하는 바와 같음. Representative analysis conditions are as follows.

HPLC 시스템 : Agilent 1290 HPLC 시스템 (Agilent Technologies) HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

검출기 : 자외 흡광도계 (측정 파장 : 280 ㎚) Detector: UV absorbance meter (measurement wavelength: 280 nm)

칼럼 : Acquity BEH Phenyl (2.1 × 50 ㎜, 1.7 ㎛, Waters 제조) Column: Acquity BEH Phenyl (2.1 × 50 mm, 1.7 μm, manufactured by Waters)

칼럼 온도 : 75 ℃ Column temperature: 75 °C

유속 : 0.8 ㎖/분Flow rate: 0.8 ml/min

샘플 주입량 : 10 ㎕ Sample injection amount: 10 μl

이동상 A : 0.1 ％ 트리플루오로아세트산 (TFA), 15 ％ 이소프로필알코올 수용액 Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution

이동상 B : 0.075 ％ TFA, 15 ％ 이소프로필알코올아세토니트릴 용액 Mobile phase B: 0.075% TFA, 15% isopropyl alcohol acetonitrile solution

그래디언트 프로그램 (이동상 B) : 14 ％ - 36 ％ (0 분 - 15 분), 36 ％ - 80 ％ (15 - 17 분), 80 ％ - 14 ％ (17 분 - 17.1 분), 14 ％ - 14 ％ (17.1 분 - 23 분) Gradient program (mobile phase B): 14% - 36% (0 min - 15 min), 36% - 80% (15 - 17 min), 80% - 14% (17 min - 17.1 min), 14% - 14% (17.1 min - 23 min)

[F-3. 데이터 해석] [F-3. data analysis]

〔F-3-1〕 약물이 결합하고 있지 않은 항체의 L 사슬 (L0) 및 H 사슬 (H0) 에 대하여, 약물이 결합한 H 사슬 (약물이 1 개 결합한 H 사슬 : H1, 약물이 2 개 결합한 H 사슬 : H2) 은, 결합한 약물의 수에 비례하여 소수성이 증가하여 유지 시간이 커지기 때문에, 원칙적으로 L0, H0, H1, H2 의 순서로 용출된다. L0 및 H0 의 유지 시간을 비교함으로써 검출 피크를 L0, H0, H1, H2 중 어느 것에 할당할 수 있다.[F-3-1] Regarding the L chain (L0) and H chain (H0) of the antibody to which the drug is not bound, the H chain to which the drug is bound (H chain with one drug bound: H1, two drugs bound) H chain: H2) is, in principle, eluted in the order of L0, H0, H1, H2 because its hydrophobicity increases in proportion to the number of bound drugs and the retention time increases. By comparing the holding times of L0 and H0, the detection peak can be assigned to any of L0, H0, H1, and H2.

〔F-3-2〕 약물 링커에 UV 흡수가 있기 때문에, 약물 링커의 결합수에 따라, H 사슬 및 약물 링커의 몰 흡광 계수를 사용하여 하기 식에 따라서 피크 면적의 보정을 실시하였다.[F-3-2] Since the drug linker has UV absorption, the peak area was corrected according to the following formula using the H chain and the molar extinction coefficient of the drug linker according to the number of bonds of the drug linker.

Figure pat00108
Figure pat00108

여기서, 각 항체에 있어서의 L 사슬 및 H 사슬의 몰 흡광 계수 (280 ㎚) 는, 공통 조작 E 에 기재한 이미 알려진 계산 방법으로 산출한 추정값을 사용하였다. 개변 항HER2 항체의 경우, L 사슬의 몰 흡광 계수로서 26213 을, H 사슬의 몰 흡광 계수로서 81478 을 사용하였다. 마찬가지로 개변 항LPS 항체의 경우, L 사슬의 몰 흡광 계수로서 27703 을, H 사슬의 몰 흡광 계수로서 85948 을 사용하였다. 약물 링커의 몰 흡광 계수 (280 ㎚) 는, SPAAC 반응에서의 콘쥬게이션인 경우에는, 콘쥬게이션 전구체의 실측값을 사용하고, 시스테인 콘쥬게이션인 경우에는, 각 콘쥬게이션 전구체를 메르캅토에탄올 또는 N-아세틸시스테인으로 반응시키고, 말레이미드기를 숙신이미드티오에테르로 변환한 화합물의 실측값을 사용하였다.Here, the estimated values calculated by the known calculation method described in Common Operation E were used for the molar extinction coefficients (280 nm) of the L chain and the H chain in each antibody. In the case of the modified anti-HER2 antibody, 26213 was used as the molar extinction coefficient of the L chain and 81478 was used as the molar extinction coefficient of the H chain. Similarly, in the case of the modified anti-LPS antibody, 27703 was used as the molar extinction coefficient of the L chain and 85948 was used as the molar extinction coefficient of the H chain. For the molar extinction coefficient (280 nm) of the drug linker, in the case of conjugation in the SPAAC reaction, the measured value of the conjugation precursor is used, and in the case of cysteine conjugation, each conjugation precursor is mixed with mercaptoethanol or N- The measured value of the compound obtained by reacting with acetylcysteine and converting the maleimide group to succinimide thioether was used.

〔F-3-3〕 피크 면적 보정값 합계에 대한 각 사슬 피크 면적비 (％) 를 하기 식에 따라서 계산하였다.[F-3-3] The peak area ratio (%) of each chain to the sum of peak area correction values was calculated according to the following formula.

Figure pat00109
Figure pat00109

〔F-3-4〕 항체-약물 콘쥬게이트에 있어서의 항체 1 분자당의 약물 평균 결합수 (DAR) 를, 하기 식에 따라서 계산하였다.[F-3-4] The average drug binding number (DAR) per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

Figure pat00110
Figure pat00110

〔F-3-5〕 항체-약물 콘쥬게이트에 있어서의 항체 농도를, 하기 식에 따라서 계산하였다.[F-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the following formula.

Figure pat00111
Figure pat00111

여기서 항체 약물 복합체의 흡광도 (280 ㎚) 는, 항체 약물 콘쥬게이트 수용액의 실측값을 사용하였다. 희석 배수는, 흡광도를 측정할 때에 항체 약물 콘쥬게이트 수용액을 몇 배로 희석했는지를 나타내며, 통상적으로 4 배 희석이다. 항체의 몰 흡광 계수 (280 ㎚) 는, 공통 조작 E 에 기재한 이미 알려진 계산 방법으로 산출한 추정값을 사용하였다. 약물 평균 결합수는〔F-3-4〕 에서 얻어진 값을 사용하였다. 약물 링커의 몰 흡광 계수 (280 ㎚) 는, SPAAC 반응에 의한 콘쥬게이션인 경우에는, 콘쥬게이션 전구체의 실측값을 사용하고, 시스테인 콘쥬게이션인 경우에는, 각 약물 링커를 메르캅토에탄올 또는 N-아세틸시스테인으로 반응시키고, 말레이미드기를 숙신이미드티오에테르로 변환한 화합물의 실측값을 사용하였다.Here, the measured value of the antibody drug conjugate aqueous solution was used for the absorbance (280 nm) of the antibody drug conjugate. The dilution factor indicates how many times the antibody-drug conjugate aqueous solution was diluted when measuring the absorbance, and is usually a 4-fold dilution. For the molar extinction coefficient (280 nm) of the antibody, an estimated value calculated by a known calculation method described in Common Operation E was used. The average number of drug bonds was the value obtained in [F-3-4]. For the molar extinction coefficient (280 nm) of the drug linker, in the case of conjugation by the SPAAC reaction, the measured value of the conjugation precursor is used, and in the case of cysteine conjugation, each drug linker is mercaptoethanol or N-acetyl. The measured value of the compound obtained by reacting with cysteine and converting the maleimide group to succinimide thioether was used.

공통 조작 G : 항체 약물 콘쥬게이트에 있어서의 항체 농도 및 항체 1 분자당의 약물 평균 결합수의 측정 (소수성 상호 작용 - 고속 액체 크로마토그래피법 : HI - HPLC) Common operation G: Measurement of antibody concentration and average number of drug bonds per antibody molecule in the antibody drug conjugate (hydrophobic interaction - high performance liquid chromatography method: HI - HPLC)

항체 약물 콘쥬게이트에 있어서의 항체 농도 및 항체 1 분자당의 약물 평균 결합수는, 전술한 공통 조작 E 및 F 에 더하여, 이하의 방법을 사용하는 고속 액체 크로마토그래피 분석에 의해 구할 수 있다.In addition to common operations E and F described above, the antibody concentration and average number of drug bonds per antibody molecule in the antibody drug conjugate can be obtained by high-performance liquid chromatography analysis using the following method.

[G-1. HPLC 분석용 샘플의 조제] [G-1. Preparation of samples for HPLC analysis]

항체 약물 콘쥬게이트 용액 (약 1 ㎎/㎖, 60 ㎕) 을 그대로 HPLC 분석에 사용하였다.The antibody drug conjugate solution (about 1 mg/ml, 60 µl) was used for HPLC analysis as it was.

[G-2. HPLC 분석] [G-2. HPLC analysis]

대표적인 분석 조건은 하기의 두가지.Representative analysis conditions are the following two.

HPLC 시스템 : SHIMADZU CBM-20A (시마즈 제작소) HPLC system: SHIMADZU CBM-20A (Shimadzu Manufacturing Co., Ltd.)

검출기 : 자외 흡광도계 (측정 파장 : 280 ㎚) Detector: UV absorbance meter (measurement wavelength: 280 nm)

칼럼 : TSK-gel Butyl-NPR (4.6 × 100 ㎜, 2.5 ㎛, TOSOH 제조) Column: TSK-gel Butyl-NPR (4.6 × 100 mm, 2.5 μm, manufactured by TOSOH)

칼럼 온도 : 25 ℃ 부근의 일정 온도 Column temperature: constant temperature around 25 ℃

이동상 A : 1.5 M 황산암모늄 함유 25 mM 인산 완충액 (pH = 7.0) Mobile phase A: 25 mM phosphate buffer containing 1.5 M ammonium sulfate (pH = 7.0)

이동상 B : 25 mM 인산 완충액 (pH = 7.0)/이소프로필알코올 혼합액 (3 : 1) Mobile phase B: 25 mM phosphate buffer (pH = 7.0)/isopropyl alcohol mixture (3:1)

유속 : 0.8 ㎖/분Flow rate: 0.8 ml/min

샘플 주입량 : 15 ㎕ Sample injection amount: 15 μl

그래디언트 프로그램 (이동상 B) : 10 ％ - 15 ％ (0 분 - 5 분), 15 ％ - 65 ％ (5 분 - 20 분) Gradient program (mobile phase B): 10% - 15% (0 min - 5 min), 15% - 65% (5 min - 20 min)

또는 or

HPLC 시스템 : SHIMADZU CBM-20A (시마즈 제작소) HPLC system: SHIMADZU CBM-20A (Shimadzu Manufacturing Co., Ltd.)

검출기 : 자외 흡광도계 (측정 파장 : 280 ㎚) Detector: UV absorbance meter (measurement wavelength: 280 nm)

칼럼 : PolyPROPYL A (4.6 × 100 ㎜, 3 ㎛, 1500 Å, PolyLC 제조) Column: PolyPROPYL A (4.6 × 100 mm, 3 μm, 1500 Å, manufactured by PolyLC)

칼럼 온도 : 40 ℃ 부근의 일정 온도 Column temperature: constant temperature around 40 ℃

이동상 A : 1.5 M 황산암모늄 함유 20 mM 인산 완충액 (pH = 7.4) Mobile phase A: 20 mM phosphate buffer containing 1.5 M ammonium sulfate (pH = 7.4)

이동상 B : 20 mM 인산 완충액 (pH = 7.4) Mobile phase B: 20 mM phosphate buffer (pH = 7.4)

유속 : 0.8 ㎖/분Flow rate: 0.8 ml/min

샘플 주입량 : 15 ㎕ Sample injection amount: 15 μl

그래디언트 프로그램 (이동상 B) : 40 ％ - 80 ％ (0 분 - 20 분) Gradient program (mobile phase B): 40% - 80% (0 min - 20 min)

[G-3. 데이터 해석] [G-3. data analysis]

〔G-3-1〕 항체에 결합한 약물의 수에 비례하여 소수성이 증가하여 유지 시간이 커지기 때문에, SPAAC 반응에 의한 콘쥬게이션인 경우에는, 원칙적으로 DAR = 0, DAR = 2, DAR = 4 의 순서로 용출된다. DAR = 0 과의 유지 시간을 비교함으로써, 검출 피크를 DAR = 2 및 DAR = 4 중 어느 하나에 할당할 수 있다. 항체나 약물 링커의 종류에 의존하여 DAR = 1 및 DAR = 3 의 피크가 검출되는 경우도 있다. 검출 피크의 DAR 은, HI-HPLC 로 당해 피크를 분획 후, 매스 스펙트럼을 측정하여 추정하는 경우도 있다. [G-3-1] Since the hydrophobicity increases in proportion to the number of drugs bound to the antibody and the retention time increases, in the case of conjugation by the SPAAC reaction, in principle, DAR = 0, DAR = 2, DAR = 4 eluted in sequence. By comparing the retention time with DAR = 0, the detection peak can be assigned to either DAR = 2 or DAR = 4. Peaks of DAR = 1 and DAR = 3 may be detected depending on the type of antibody or drug linker. The DAR of the detection peak may be estimated by measuring the mass spectrum after fractionating the peak by HI-HPLC.

〔G-3-2〕 약물 링커에 UV 흡수가 있기 때문에, 약물 링커의 결합수에 따라, 항체 및 약물 링커의 몰 흡광 계수를 사용하여 하기 식에 따라서 피크 면적값의 보정을 실시하였다.[G-3-2] Since the drug linker has UV absorption, the peak area value was corrected according to the following equation using the molar extinction coefficients of the antibody and drug linker according to the number of drug linker bonds.

Figure pat00112
Figure pat00112

여기서, 항체의 몰 흡광 계수 (280 ㎚) 는, 공통 조작 E 에 기재한 이미 알려진 계산 방법으로 산출한 추정값을 사용하였다. 약물 링커의 몰 흡광 계수 (280 ㎚) 는, 콘쥬게이션 전구체의 실측값을 사용하였다.Here, as the molar extinction coefficient (280 nm) of the antibody, an estimated value calculated by a known calculation method described in Common Operation E was used. The measured value of the conjugated precursor was used as the molar extinction coefficient (280 nm) of the drug linker.

〔G-3-3〕 피크 면적 보정값 합계에 대한 항체 피크 면적비 (％) 를 하기 식에 따라서 계산하였다.[G-3-3] The antibody peak area ratio (%) to the sum of peak area correction values was calculated according to the following formula.

Figure pat00113
Figure pat00113

〔G-3-4〕 항체-약물 콘쥬게이트에 있어서의 항체 1 분자당의 약물 평균 결합수를, 하기 식에 따라서 계산하였다.[G-3-4] The average number of drug bonds per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

Figure pat00114
Figure pat00114

〔G-3-5〕 항체-약물 콘쥬게이트에 있어서의 항체 농도는,〔F-3-5〕 에 기재된 식에 따라서 계산하였다. 그 때, 약물 평균 결합수는〔G-3-4〕 에서 얻어진 값을 사용하였다.[G-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the formula described in [F-3-5]. At that time, the value obtained in [G-3-4] was used as the average number of drug bonds.

본 발명의 신규 CDN 유도체, 항체 약물 콘쥬게이트 또는 그들 제조 중간체에는, 입체 이성체 혹은 부제 탄소 원자에서 유래하는 광학 이성체, 기하 이성체, 호변 이성체 또는 d 체, l 체, 아트로프 이성체 등의 광학 이성체가 존재하는 경우도 있지만, 이들 이성체, 광학 이성체 및 이들 혼합물 모두 본 발명에 포함된다.In the novel CDN derivatives, antibody drug conjugates or intermediates produced therefrom of the present invention, stereoisomers or optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers exist. In some cases, these isomers, optical isomers, and mixtures thereof are all included in the present invention.

본 발명의 항체 약물 콘쥬게이트에 있어서, 항체 1 분자에 대한 약물의 결합수는, 그 유효성, 안전성에 영향을 미치는 중요 인자이다. 항체 약물 콘쥬게이트의 제조는, 약물의 결합수가 일정한 수가 되도록, 반응시키는 원료·시약의 사용량 등의 반응 조건을 규정하여 실시되지만, 저분자 화합물의 화학 반응과는 달리, 상이한 수의 약물이 결합한 혼합물로서 얻어지는 것이 통상적이다. 항체 1 분자에 대한 약물의 결합수는 평균값, 즉, 평균 약물 결합수 (DAR) 로서 특정할 수 있다. 항체 분자에 대한 고리형 디뉴클레오티드 유도체의 결합수는 컨트롤 가능하고, 1 항체당의 약물 평균 결합수로서, 1 내지 10 의 범위의 고리형 디뉴클레오티드 유도체를 결합시킬 수 있지만, 바람직하게는 1 내지 8 개이며, 보다 바람직하게는 1 내지 5 개이다.In the antibody drug conjugate of the present invention, the number of drug bonds to one antibody molecule is an important factor affecting its effectiveness and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions such as the amount of raw materials and reagents to be reacted so that the number of drugs bound is a certain number, but unlike the chemical reaction of low-molecular compounds, as a mixture in which different numbers of drugs are bound. It is usual to obtain The number of drugs bound to one antibody molecule can be determined as an average value, that is, the average number of drug bonds (DAR). The number of bindings of cyclic dinucleotide derivatives to antibody molecules can be controlled, and the average number of drug bindings per antibody ranges from 1 to 10, but preferably 1 to 8. and more preferably 1 to 5.

본 발명의 항체 약물 콘쥬게이트에 있어서, 항체 Ab 가, 항체 Ab 의 리모델링 된 당사슬로부터 L 에 결합하고 있는 경우, 항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 약물 결합수 m2 는 1 또는 2 의 정수이다. 당해 당사슬이 N297 당사슬이고, 당사슬이 N297-(Fuc)SG 인 경우, m2 는 2 이고, DAR 은 3 ∼ 5 의 범위 (바람직하게는, 3.2 ∼ 4.8 의 범위이며, 보다 바람직하게는, 3.5 ∼ 4.2 의 범위) 이다. N297 당사슬이 N297-(Fuc)MSG1, N297-(Fuc)MSG2 또는 N297-(Fuc)MSG1 과 N297-(Fuc)MSG2 의 혼합물인 경우, m2 는 1 이고, DAR 은 1 ∼ 3 의 범위 (바람직하게는, 1.0 ∼ 2.5 의 범위, 보다 바람직하게는, 1.2 ∼ 2.2 의 범위) 이다.In the antibody drug conjugate of the present invention, when antibody Ab is bound to L from the remodeled sugar chain of antibody Ab, the number m 2 of drug binding per antibody molecule in the antibody drug conjugate is an integer of 1 or 2 am. When the sugar chain is N297 sugar chain and the sugar chain is N297-(Fuc)SG, m 2 is 2 and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.8). scope of 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2 or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1 and DAR is in the range of 1 to 3 (preferably preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

또한, 당업자이면 본원의 실시예의 기재로부터 항체에 필요한 수의 약물을 결합시키는 반응을 설계할 수 있고, 고리형 디뉴클레오티드 유도체의 결합수를 컨트롤 한 항체를 취득할 수 있다.In addition, those skilled in the art can design a reaction in which a necessary number of drugs are bound to an antibody based on the description of the examples herein, and an antibody in which the number of bound cyclic dinucleotide derivatives is controlled can be obtained.

또한, 본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체는, 대기 중에 방치하거나, 또는 재결정함으로써, 수분을 흡수하여, 흡착수가 묻거나, 수화물이 되는 경우가 있으며, 그와 같은 물을 포함하는 화합물 및 염도 본 발명에 포함된다.In addition, the CDN derivatives, antibody drug conjugates, and intermediates for their production of the present invention may absorb moisture when left in the air or recrystallized, and may become stained with adsorbed water or become hydrates, including such water. Compounds and salts which are also included in the present invention.

본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체가, 아미노기 등의 염기성 기를 갖는 경우, 원하는 바에 따라 의약적으로 허용되는 염으로 할 수 있다. 그러한 염으로는, 예를 들어 염산염, 요오드화수소산염 등의 할로겐 화수소산염 ; 질산염, 과염소산염, 황산염, 인산염 등의 무기산염 ; 메탄술폰산염, 트리플루오로메탄술폰산염, 에탄술폰산염 등의 저급 알칸술폰산염 ; 벤젠술폰산염, p-톨루엔술폰산염 등의 아릴술폰산염 ; 포름산, 아세트산, 말산, 푸마르산염, 숙신산염, 시트르산염, 타르타르산염, 옥살산염, 말레산염 등의 유기산염 ; 및 오르니틴산염, 글루타민산염, 아스파르트산염 등의 아미노산염을 들 수 있다.When the CDN derivative of the present invention, the antibody drug conjugate, or their production intermediate has a basic group such as an amino group, it can be used as a pharmaceutically acceptable salt as desired. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide; Inorganic acid salts, such as a nitrate, a perchlorate, a sulfate, and a phosphate; lower alkanesulfonic acid salts such as methanesulfonic acid salts, trifluoromethanesulfonic acid salts, and ethanesulfonic acid salts; aryl sulfonic acid salts such as benzene sulfonic acid salt and p-toluene sulfonic acid salt; organic acid salts such as formic acid, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as ornitrate, glutamate, and aspartate.

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트는, 인산기 및/또는 티오 인산기를 그 구조에 포함하기 때문에, 일반적으로 염기 부가 염을 형성하는 것이 가능하다. 또, 그들의 제조 중간체가, 카르복시기 등의 산성기를 갖는 경우에도, 일반적으로 염기 부가 염을 형성하는 것이 가능하다. 의약적으로 허용되는 염으로는, 예를 들어, 나트륨염, 칼륨염, 리튬염 등의 알칼리 금속염 ; 칼슘염, 마그네슘염 등의 알칼리 토금속염 ; 암모늄염 등의 무기염 ; 디벤질아민염, 모르폴린염, 페닐글리신알킬에스테르염, 에틸렌디아민염, N-메틸글루카민염, 디에틸아민염, 트리에틸아민염, 시클로헥실아민염, 디시클로헥실아민염, N,N'-디벤질에틸렌디아민염, 디에탄올아민염, N-벤질-N-(2-페닐에톡시)아민염, 피페라진염, 테트라메틸암모늄염, 트리스(하이드록시메틸)아미노메탄염 등의 유기 아민염, 등을 들 수 있다.Since the CDN derivative or antibody drug conjugate of the present invention contains a phosphate group and/or a thiophosphate group in its structure, it is generally possible to form a base addition salt. In addition, even when these production intermediates have an acidic group such as a carboxy group, it is generally possible to form a base addition salt. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; Inorganic salts, such as an ammonium salt; Dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N Organic amines such as '-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt Salt, etc. are mentioned.

본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체는, 공기중의 수분을 흡수하는 것 등에 의해 수화물로서 존재하는 경우도 있다. 본 발명의 용매화물로는, 의약적으로 허용할 수 있는 것이면 특별히 한정되지 않지만, 구체적으로는, 수화물, 에탄올화물, 2-프로판올화물 등이 바람직하다. 또, 본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체 중에 질소 원자가 존재하는 경우에는 N-옥사이드체로 되어 있어도 되고, 이들 용매화물 및 N-옥사이드체도 본 발명의 범위에 포함된다. 또, 본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체 중에 황 원자가 존재하는 경우에는 술폭시드체로 되어 있어도 되며, 이들 용매화물 및 술폭시드체도 본 발명의 범위에 포함된다.The CDN derivatives, antibody drug conjugates, and their manufacturing intermediates of the present invention may exist as hydrates by absorbing moisture in the air or the like. The solvate of the present invention is not particularly limited as long as it is pharmaceutically acceptable, but specifically, hydrates, ethanol compounds, 2-propanol compounds and the like are preferred. In addition, when a nitrogen atom is present in the CDN derivative, antibody drug conjugate, and intermediates for their production of the present invention, they may be N-oxides, and these solvates and N-oxides are also included in the scope of the present invention. In addition, when a sulfur atom is present in the CDN derivative, antibody drug conjugate, and intermediates for their production of the present invention, they may be sulfoxide compounds, and these solvates and sulfoxide compounds are also included in the scope of the present invention.

또, 본 발명에는, 각종 방사성 또는 비방사성 동위체로 라벨된 화합물도 포함된다. 본 발명의 CDN 유도체, 항체 약물 콘쥬게이트, 그들의 제조 중간체를 구성하는 원자의 1 이상에, 원자 동위체의 비천연 비율도 함유할 수 있다. 원자 동위체로는, 예를 들어, 중수소 (2H), 트리튬 (3H), 요오드-125 (125I) 또는 탄소-14 (14C) 등을 들 수 있다. 또, 본 발명 화합물은, 예를 들어, 트리튬 (3H), 요오드-125 (125I) 또는 탄소-14 (14C) 와 같은 방사성 동위체로 방사성 표지될 수 있다. 방사성 표지된 화합물은, 치료 또는 예방제, 연구 시약, 예를 들어, 어세이 시약, 및 진단제, 예를 들어, 인비보 화상 진단제로서 유용하다. 본 발명의 항체 약물 콘쥬게이트의 모든 동위체 변이종은, 방사성 유무를 묻지 않고, 본 발명의 범위에 포함된다.Moreover, compounds labeled with various radioactive or non-radioactive isotopes are also included in the present invention. At least one atom constituting the CDN derivatives, antibody drug conjugates, and intermediates thereof of the present invention may also contain unnatural proportions of atomic isotopes. As an atomic isotope, deuterium (2H), tritium (3H), iodine-125 (125I), or carbon-14 (14C) etc. are mentioned, for example. In addition, the compound of the present invention may be radiolabeled with a radioactive isotope such as, for example, tritium (3H), iodine-125 (125I) or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, such as assay reagents, and diagnostic agents, such as in vivo imaging agents. All isotopic variants of the antibody drug conjugate of the present invention are included in the scope of the present invention, regardless of whether or not they are radioactive.

＜4. 의약＞ <4. Medicines>

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트는, 암세포에 대하여 항종양 면역 활성 또는 세포 상해 활성을 나타내기 때문에, 의약으로서, 특히 암에 대한 치료제 및/또는 예방제, 또는 항종양제로서 사용할 수 있다.Since the CDN derivative or antibody drug conjugate of the present invention exhibits antitumor immune activity or cytotoxic activity against cancer cells, it can be used as a medicine, particularly as a therapeutic and/or preventive agent for cancer, or as an antitumor agent.

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트가 적용되는 암의 종류로는, 폐암 (비소세포 폐암, 소세포 폐암 등), 신장암, 요로 상피암, 대장암, 전립선암, 다형 신경교아종, 난소암 (표층 상피성 종양, 간질성 종양, 배세포 종양 등), 췌암, 유방암, 멜라노마, 간암, 방광암, 위암, 식도암, 자궁체암, 정소암 (세미노마, 비세미노마), 자궁경암, 태반 융모암, 다형 신경교아종, 뇌종양, 두경부암, 갑상선암, 중피종, 소화관 간질 종양 (Gastrointestinal Stromal Tumor, GIST), 담낭암, 담관암, 부신암, 유극세포암, 백혈병, 악성 림프종, 형질 세포종, 골수종, 육종 등을 들 수 있지만, 항체 약물 콘쥬게이트에 대해서는, 치료 대상이 되는 암세포에 있어서 항체 약물 콘쥬게이트 중의 항체를 인식할 수 있는 단백질을 발현하고 있는 암세포이면 이들에는 한정되지 않는다.Types of cancer to which the CDN derivative or antibody drug conjugate of the present invention is applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), kidney cancer, urinary tract epithelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (superficial layer epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine body cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, Glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, angular cell cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, etc. However, the antibody drug conjugate is not limited to these, as long as the cancer cells to be treated express a protein capable of recognizing the antibody in the antibody drug conjugate.

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트는, 포유 동물에 대하여 적합하게 투여할 수 있지만, 보다 바람직하게는 인간이다.The CDN derivative or antibody drug conjugate of the present invention can be suitably administered to mammals, but more preferably to humans.

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트가 포함되는 의약 조성물에 있어서 사용되는 물질로는, 투여량이나 투여 농도에 있어서, 이 분야에 있어서 통상적으로 사용되는 제제 첨가물 그 외로부터 적절히 선택하여 적용할 수 있다.As the substance used in the pharmaceutical composition containing the CDN derivative or antibody drug conjugate of the present invention, it can be appropriately selected and applied from formulation additives and others commonly used in the field in terms of dosage or administration concentration. there is.

본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트는, 1 종 이상의 약학적으로 적합성의 성분을 포함하는 의약 조성물로서 투여될 수 있다. 예를 들어, 상기 의약 조성물은, 대표적으로는, 1 종 이상의 약학적 캐리어 (예를 들어, 멸균한 액체 (예를 들어, 물 및 기름 (석유, 동물, 식물, 또는 합성 기원의 기름 (예를 들어, 낙화생유, 대두유, 광유, 참기름 등)) 을 포함한다)) 를 포함한다. 물은, 상기 의약 조성물이 정맥내 투여되는 경우에, 보다 대표적인 캐리어이다. 식염수 용액, 그리고 덱스트로스 수용액 및 글리세롤 수용액도 또한, 액체 캐리어로서, 특히, 주사용 용액을 위해서 사용될 수 있다. 적절한 약학적 부형제는, 당해 분야에서 공지이다. 상기 조성물은 또한, 원한다면, 미량의 습윤제 혹은 유화제, 또는 pH 완충화제를 포함할 수 있다. 적절한 약학적 캐리어의 예는, E. W. Martin 에 의한 「Remington's Pharmaceutical Sciences」 에 기재된다. 그 처방은, 투여의 양태에 대응한다.The CDN derivative or antibody drug conjugate of the present invention can be administered as a pharmaceutical composition containing one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically comprises one or more pharmaceutical carriers (e.g., sterilized liquid (e.g., water and oil (petroleum, animal, plant, or oil of synthetic origin)) For example, including peanut oil, soybean oil, mineral oil, sesame oil, etc.)))). Water is a more representative carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextrose and glycerol may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition may also contain trace amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The prescription corresponds to the mode of administration.

여러 가지 송달 시스템이 공지이며, 본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트를 투여하기 위해서 사용될 수 있다. 도입 방법으로는, 피내, 근육내, 복강내, 정맥내, 및 피하의 경로를 들 수 있지만, 이들에 한정되지 않는다. 투여는, 예를 들어, 주입 또는 볼러스 주사에 의한 것일 수 있다. 특정한 바람직한 실시형태에 있어서, 상기 CDN 유도체 또는 항체 약물 콘쥬게이트의 투여는, 주입에 의한 것이다. 비경구적 투여는, 바람직한 투여 경로이다.Several delivery systems are known and can be used to administer the CDN derivative or antibody drug conjugate of the present invention. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, administration of the CDN derivative or antibody drug conjugate is by infusion. Parenteral administration is a preferred route of administration.

대표적 실시형태에 있어서, 상기 항체 약물 콘쥬게이트를 포함하는 의약 조성물은, 인간에 대한 정맥내 투여에 적합한 의약 조성물로서, 상습적 순서에 따라서 처방된다. 대표적으로는, 정맥내 투여를 위한 조성물은, 멸균 등장성의 수성 완충액 중의 용액이다. 필요한 경우, 상기 의약은 또한, 가용화제 및 주사 부위에서의 동통을 완화시키기 위한 국소 마취제 (예를 들어, 리그노카인) 를 포함할 수 있다. 일반적으로, 상기 성분은, 예를 들어, 활성제의 양을 나타내는 앰플 또는 사셰 등의 밀봉하여 시일된 용기 중의 건조 동결 건조 분말 또는 무수의 농축물로서, 별개로, 또는 단위 제형 중에서 함께 혼합하여, 중 어느 것으로 공급된다. 상기 의약 조성물이 주입에 의해 투여될 예정인 경우, 그것은, 예를 들어, 멸균의 제약 그레이드의 물 또는 식염수를 포함하는 주입 보틀로 투약될 수 있다. 상기 의약이 주사에 의해 투여되는 경우, 주사용 멸균수 또는 식염수의 앰플은, 예를 들어, 상기 성분이 투여 전에 혼합될 수 있도록, 제공될 수 있다. 상기 의약 조성물은, 용액으로서 제공되는 경우도 있다.In a representative embodiment, the pharmaceutical composition containing the antibody drug conjugate is a pharmaceutical composition suitable for intravenous administration to humans, and is prescribed according to a customary procedure. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also contain a solubilizing agent and a local anesthetic (eg, lignocaine) to relieve pain at the injection site. Generally, the ingredients are either separately as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent, or mixed together in unit dosage form, in a medium dosage form. which is supplied When the pharmaceutical composition is to be administered by infusion, it may be administered, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline may be provided, for example, so that the components may be mixed prior to administration. The pharmaceutical composition may be provided as a solution.

본 발명의 의약 조성물에는 본원의 CDN 유도체 또는 항체 약물 콘쥬게이트만을 포함하는 의약 조성물이어도 되고, CDN 유도체 또는 항체 약물 콘쥬게이트 및 적어도 하나의 이것 이외의 암 치료제를 포함하는 의약 조성물이어도 된다. 본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트는, 다른 암 치료제와 함께 투여할 수도 있으며, 이에 따라 항암 효과를 증강시킬 수 있다. 이와 같은 목적으로 사용되는 다른 항암제는, CDN 유도체 또는 항체 약물 콘쥬게이트와 동시에, 따로 따로, 혹은 연속해서 개체에 투여되어도 되고, 각각의 투여 간격을 바꾸어 투여해도 된다. 이와 같은 암 치료제로서, abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin 또는 국제 공개 제WO2003/038043호 팜플렛에 기재된 약제, 또한 LH-RH 아날로그 (류프로렐린, 고세렐린 등), 에스트라무스틴·포스페이트, 에스트로겐 길항약 (타목시펜, 라록시펜 등), 아로마타제 저해제 (아나스트로졸, 레트로졸, 엑세메스탄 등), 면역 체크 포인트 저해약 (니보르맙, 이필리뮤맙 등) 등을 들 수 있지만, 항종양 활성을 갖는 약제이면 한정되지는 않는다.The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the CDN derivative or antibody drug conjugate of the present application, or a pharmaceutical composition containing the CDN derivative or antibody drug conjugate and at least one other cancer therapeutic agent. The CDN derivative or antibody drug conjugate of the present invention may be administered together with other cancer therapeutic agents, thereby enhancing anticancer effects. Other anticancer agents used for this purpose may be administered to the subject simultaneously, separately, or continuously with the CDN derivative or antibody drug conjugate, or may be administered at different intervals between administrations. As such a cancer therapeutic agent, abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin or a drug described in the pamphlet of International Publication No. WO2003/038043, also an LH-RH analog (leuprorelin , goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), immune checkpoint inhibitors (nivorab , ipilimumab, etc.), but it is not limited as long as it is a drug having antitumor activity.

이와 같은 의약 조성물은, 선택된 조성과 필요한 순도를 갖는 제제로서, 동결 건조 제제 혹은 액상 제제로서 제제화하면 된다. 동결 건조 제제로서 제제화할 때, 이 분야에 있어서 사용되는 적당한 제제 첨가물이 포함되는 제제여도 된다. 또 액제에 있어서도 동일하게 하여, 이 분야에 있어서 사용되는 각종 제제 첨가물을 포함하는 액상 제제로서 제제화할 수 있다.Such a pharmaceutical composition may be formulated as a freeze-dried preparation or liquid preparation as a preparation having a selected composition and required purity. When formulated as a freeze-dried preparation, a preparation containing suitable additives used in this field may be used. Also, liquid preparations can be formulated as liquid preparations containing various preparation additives used in this field in the same manner.

의약 조성물의 조성 및 농도는 투여 방법에 따라서도 변화하지만, 본 발명의 의약 조성물에 포함되는 항체 약물 콘쥬게이트는, 항체 약물 콘쥬게이트의 항원에 대한 친화성, 즉, 항원에 대한 해리 정수 (定數) (Kd 값) 의 점에 있어서, 친화성이 높을 (Kd 값이 낮을) 수록, 소량의 투여량이더라도 약효를 발휘시킬 수 있다. 따라서, 항체 약물 콘쥬게이트의 투여량의 결정에 있어서는, 항체 약물 콘쥬게이트와 항원의 친화성의 상황에 기초하여 투여량을 설정할 수도 있다. 본 발명의 CDN 유도체 또는 항체 약물 콘쥬게이트를 인간에 대하여 투여할 때에는, 예를 들어, 약 0.001 ∼ 100 ㎎/㎏ 을 1 회 혹은 1 ∼ 180 일간에 1 회의 간격으로 복수 회 투여하면 된다.The composition and concentration of the pharmaceutical composition vary depending on the administration method, but the antibody drug conjugate contained in the pharmaceutical composition of the present invention has an affinity for the antigen of the antibody drug conjugate, that is, a dissociation constant for the antigen. ) (Kd value), the higher the affinity (the lower the Kd value), the more the drug effect can be exhibited even at a small dose. Therefore, in determining the dose of the antibody drug conjugate, the dose can be set based on the affinity between the antibody drug conjugate and the antigen. When administering the CDN derivative or antibody drug conjugate of the present invention to humans, for example, about 0.001 to 100 mg/kg may be administered once or multiple times at intervals of 1 to 180 days.

이하 본 발명을 실시예에서 설명하지만 본 발명은 이들에 한정되는 것은 아니다.Hereinafter, the present invention will be described in examples, but the present invention is not limited thereto.

실시예Example

이하의 실시예에 있어서, 실온은 15 ℃ 내지 35 ℃ 를 나타낸다. 탈수 아세토니트릴은, 칸토 화학에서 판매되고 있는 아세토니트릴 (탈수) -Super- 또는 와코 쥰야쿠 공업에서 판매되고 있는 아세토니트릴 (초탈수) 을 사용하였다. 피리딘은, 칸토 화학에서 판매되고 있는 피리딘 (탈수) -Super- 를 사용하였다. 실리카 겔 크로마토그래피는, Biotage SNAP Ultra (Biotage 제조), Chromatorex Q-Pack SI (후지 실리시아 제조) 또는 Purif-Pack-Ex SI (쇼코 사이언스 제조) 를 사용하여 실시하였다. DIOL 실리카 겔 칼럼 크로마토그래피는, Chromatorex Q-pack DIOL (후지 실리시아 제조) 을 사용하여 실시하였다. C18 실리카 겔 칼럼 크로마토그래피는, Biotage SNAP Ultra C18 (Biotage 제조) 을 사용하여 실시하였다. 아미노 실리카 겔 칼럼 크로마토그래피는, Biotage SNAP Isolute NH2 (Biotage 제조) 를 사용하여 실시하였다. 분취 HPLC 는, SHIMADZU SPD-M10A HPLC 시스템 (시마즈 제작소) 등을 사용하여 실시하였다. 분취 칼럼은, Kinetex (5 ㎛, C18, 100 Å, 250 × 30.0 ㎜, Phenomenex 제조) 또는 Kinetex (5 ㎛, C18, 100 Å, 250 × 21.2 ㎜, Phenomenex 제조) 를 사용하였다.In the examples below, room temperature indicates 15°C to 35°C. As the dehydrated acetonitrile, acetonitrile (dehydrated) -Super- sold by Kanto Chemical or acetonitrile (super dehydrated) sold by Wako Pure Chemical Industries was used. As pyridine, pyridine (dehydrated) -Super- sold by Kanto Chemical was used. Silica gel chromatography was performed using Biotage SNAP Ultra (manufactured by Biotage), Chromatorex Q-Pack SI (manufactured by Fuji Silysia) or Purif-Pack-Ex SI (manufactured by Shoko Science). DIOL silica gel column chromatography was performed using Chromatorex Q-pack DIOL (manufactured by Fuji Silysia). C18 silica gel column chromatography was performed using Biotage SNAP Ultra C18 (manufactured by Biotage). Amino silica gel column chromatography was performed using Biotage SNAP Isolute NH 2 (manufactured by Biotage). Preparative HPLC was performed using a SHIMADZU SPD-M10A HPLC system (Shimadzu Corporation) or the like. As the preparative column, Kinetex (5 μm, C18, 100 Å, 250 × 30.0 mm, manufactured by Phenomenex) or Kinetex (5 μm, C18, 100 Å, 250 × 21.2 mm, manufactured by Phenomenex) was used.

각종 스펙트럼 데이터의 측정에는 이하의 기기를 사용하였다. 1H-NMR 스펙트럼은, JEOL ECS-400 (400 ㎒), Varian 400-MR (400 ㎒) 또는 Varian Unity Inova 500 (500 ㎒) 을 사용하여 측정하였다. 31P-NMR 스펙트럼은, JEOL ECS-400 (160 ㎒) 을 사용하여 측정하였다. 매스 스펙트럼은, Agilent 6130 Quadrupole LC/MS 시스템 (Agilent Technologies) 을 사용하여 측정하였다. LC/MS 의 측정은, 이하의 조건으로 실시한 [칼럼 : Develosil Combi-RP, 5 ㎛, 50 × 2.0 ㎜ (노무라 화학 제조), 이동상 : 0.1 ％ 포름산아세토니트릴 용액/0.1 ％ 포름산 수용액, 0.1 ％ 포름산아세토니트릴 용액 : 2 ％ - 100 ％ (0 분 - 5 분 또는 0 분 -10 분)].The following instruments were used to measure various spectral data. 1 H-NMR spectrum was measured using JEOL ECS-400 (400 MHz), Varian 400-MR (400 MHz) or Varian Unity Inova 500 (500 MHz). 31 P-NMR spectrum was measured using JEOL ECS-400 (160 MHz). Mass spectra were measured using an Agilent 6130 Quadrupole LC/MS system (Agilent Technologies). LC/MS was measured under the following conditions [Column: Develosil Combi-RP, 5 μm, 50 × 2.0 mm (manufactured by Nomura Chemical), mobile phase: 0.1% formic acid acetonitrile solution/0.1% formic acid aqueous solution, 0.1% formic acid Acetonitrile solution: 2% - 100% (0 min - 5 min or 0 min -10 min)].

실시예 1 : CDN1 의 합성 Example 1: Synthesis of CDN1

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) )-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5, 8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 108][Formula 108]

Figure pat00115
Figure pat00115

[합성 스킴][Synthesis Scheme]

[화학식 109][Formula 109]

Figure pat00116
Figure pat00116

(공정 1) (Process 1)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-요오드-7H-피롤로[2,3-d]피리미딘-4-아민7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-iodo-7H- Pyrrolo[2,3-d]pyrimidin-4-amine

이미 알려진 문헌 (Tetrahedron 2007, 63, 9850-9861) 의 5-요오드투베르시딘 (1.0 g) 의 N,N-디메틸포름아미드 (10 ㎖) 용액에, 0 ℃ 에서 디-tert-부틸실릴비스(트리플루오로메탄술포네이트) (1.24 ㎖) 를 천천히 적하한 후, 동 (同) 온도에서 30 분간 교반하였다. 이미다졸 (868 ㎎) 을 0 ℃ 에서 첨가한 후, 실온까지 승온하여 30 분간 교반하였다. 실온에서 tert-부틸디메틸클로로실란을 첨가하고, 동 온도에서 밤새 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (910 ㎎) 을 얻었다. In a solution of 5-iodotubercidine (1.0 g) in N, N-dimethylformamide (10 ml) of the previously known literature (Tetrahedron 2007, 63, 9850-9861), di-tert-butylsilylbis at 0 ° C. (Trifluoromethanesulfonate) (1.24 ml) was slowly added dropwise thereto, followed by stirring at the same temperature for 30 minutes. After adding imidazole (868 mg) at 0°C, the temperature was raised to room temperature and stirred for 30 minutes. tert-butyldimethylchlorosilane was added at room temperature and stirred at the same temperature overnight. After stopping the reaction by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (910 mg).

Figure pat00117
Figure pat00117

(공정 2) (Process 2)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(3,3-디에톡시프로파-1-인-1-일)-7H-피롤로[2,3-d]피리미딘-4-아민 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(3,3 -diethoxypropa-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 1 에서 얻어진 화합물 (910 ㎎) 의 N,N-디메틸포름아미드 (3.0 ㎖) - 테트라하이드로푸란 (9.0 ㎖) 혼합 용액에 프로파르길알데히드디메틸아세탈 (1.01 ㎖), 트리에틸아민 (0.392 ㎖), 테트라키스(트리페닐포스핀)팔라듐(0) (163 ㎎), 및 요오드화구리(I) (53.6 ㎎) 을 차례로 첨가하고, 40 ℃ 에서 18 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 아세트산에틸을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (878 ㎎) 을 얻었다. Propargylaldehyde dimethyl acetal (1.01 ml) and triethylamine (0.392 ml) were added to a mixed solution of N, N-dimethylformamide (3.0 ml) - tetrahydrofuran (9.0 ml) of the compound (910 mg) obtained in step 1 above. ), tetrakis(triphenylphosphine)palladium(0) (163 mg), and copper(I) iodide (53.6 mg) were added in this order, and the mixture was stirred at 40°C for 18 hours. A saturated aqueous solution of sodium hydrogen carbonate and ethyl acetate were added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (878 mg).

Figure pat00118
Figure pat00118

(공정 3) (Process 3)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 2 에서 얻어진 화합물 (878 ㎎) 의 에탄올 (8.8 ㎖) 용액에 10 ％ 팔라듐탄소 (M) wet (500 ㎎) 를 첨가하고, 수소 분위기하, 실온에서 9 시간 교반하였다. 촉매를 여과 제거한 후, 디클로로메탄으로 세정하고, 여과액을 감압 농축하였다. 잔류물의 아세트산 (8.8 ㎖) 용액에 10 ％ 팔라듐탄소 (M) wet (500 ㎎) 를 첨가하고, 수소 분위기하, 40 ℃ 에서 2 일간 교반하였다. 촉매를 여과 제거한 후, 디클로로메탄으로 세정하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (603 ㎎) 을 얻었다. 10% palladium carbon (M) wet (500 mg) was added to a solution of the compound (878 mg) obtained in step 2 in ethanol (8.8 ml), and the mixture was stirred at room temperature under a hydrogen atmosphere for 9 hours. After filtering off the catalyst, it was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. 10% palladium carbon (M) wet (500 mg) was added to a solution of the residue in acetic acid (8.8 ml), and the mixture was stirred under a hydrogen atmosphere at 40°C for 2 days. After filtering off the catalyst, it was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (603 mg).

Figure pat00119
Figure pat00119

(공정 4) (Process 4)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6, 7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 3 에서 얻어진 화합물 (2.17 g) 의 디클로로메탄 (21.7 ㎖) 용액에 실온에서, 피리딘 (1.56 ㎖), N,N-디메틸아미노피리딘 (94.5 ㎎), 및 염화벤조일 (0.898 ㎖) 을 차례로 첨가하고, 50 ℃ 에서 15 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시켰다. 디클로로메탄으로 추출 후, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.91 g) 을 얻었다. To a solution of the compound (2.17 g) obtained in step 3 above in dichloromethane (21.7 ml) at room temperature, pyridine (1.56 ml), N,N-dimethylaminopyridine (94.5 mg), and benzoyl chloride (0.898 ml) were sequentially added. And stirred at 50 degreeC for 15 hours. The reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution. After extraction with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (1.91 g).

Figure pat00120
Figure pat00120

(공정 5) (Process 5)

6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}- 6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (1.91 g) 의 디클로로메탄 (15 ㎖) 용액에, 0 ℃ 에서 조제한 불화수소-피리딘 (0.30 ㎖) 과 피리딘 (1.88 ㎖) 의 혼합액을 첨가하고, 0 ℃ 에서 2 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시켰다. 반응액을 디클로로메탄으로 추출한 후, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 피리딘 (15 ㎖) 에 용해하고, 4,4'-디메톡시트리틸클로라이드 (1.17 g) 를 첨가하고, 0 ℃ 에서 12 시간 교반하였다. 메탄올을 첨가하고 30 분간 교반한 후, 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시켰다. 반응액을 디클로로메탄으로 추출한 후, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.98 g) 을 얻었다. To a dichloromethane (15 ml) solution of the compound (1.91 g) obtained in step 4 above, a mixture of hydrogen fluoride-pyridine (0.30 ml) and pyridine (1.88 ml) prepared at 0°C was added, and the mixture was stirred at 0°C for 2 hours. did The reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution. After the reaction solution was extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (15 ml), 4,4'-dimethoxytrityl chloride (1.17 g) was added, and the mixture was stirred at 0 DEG C for 12 hours. After adding methanol and stirring for 30 minutes, the reaction was stopped by adding saturated aqueous sodium hydrogen carbonate solution. After the reaction solution was extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (1.98 g).

Figure pat00121
Figure pat00121

(공정 6) (Process 6)

6-벤조일-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetra azabenzo[cd]azulene

상기 공정 5 에서 얻어진 화합물 (1.98 g) 의 디클로로메탄 (23.9 ㎖) 용액에 N,N-디이소프로필에틸아민 (1.02 ㎖) 과 2-시아노에틸N,N-디이소프로필클로로포스포로아미다이트 (1.07 ㎖) 를 첨가하고, 실온에서 15 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시켰다. 반응액을 디클로로메탄으로 추출 후, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.06 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 7 : 3) 로서 얻었다. N, N-diisopropylethylamine (1.02 ml) and 2-cyanoethyl N, N-diisopropylchlorophosphoroamide in dichloromethane (23.9 ml) solution of the compound (1.98 g) obtained in step 5 above (1.07 ml) was added, and the mixture was stirred at room temperature for 15 hours. The reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution. After extracting the reaction solution with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (2.06 g) as a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 7:3).

Figure pat00122
Figure pat00122

(공정 7) (Process 7)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6 ,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 6 에서 얻어진 화합물 (1.37 g) 의 아세토니트릴 (6.67 ㎖) 용액에 물 (48 ㎕) 과 트리플루오로아세트산피리딘염 (335 ㎎) 을 첨가하고, 실온에서 15 분간 교반하였다. 반응액에 tert-부틸아민 (6.67 ㎖) 을 첨가하고, 실온에서 15 분간 교반하였다. 반응액을 감압 농축한 후, 잔류물을 아세토니트릴 (5 ㎖) 로 2 회 공비하였다. 잔류물의 디클로로메탄 (16.7 ㎖) 용액에 물 (0.240 ㎖) 을 첨가한 후, 디클로로아세트산 (0.953 ㎖) 의 디클로로메탄 (16.7 ㎖) 용액을 첨가하고, 실온에서 15 분간 교반하였다. 피리딘 (1.82 ㎖) 을 첨가하여 반응을 정지시킨 후, 반응액을 감압 농축하였다. 잔류물을 탈수 아세토니트릴 (10 ㎖) 로 3 회 공비하고, 마지막 1 회는 5 ㎖ 정도의 아세토니트릴을 남겼다. 얻어진 표제 화합물의 아세토니트릴 용액을 그대로 다음의 반응에 사용하였다.Water (48 µl) and trifluoroacetic acid pyridine salt (335 mg) were added to a solution of the compound (1.37 g) obtained in step 6 in acetonitrile (6.67 ml), and the mixture was stirred at room temperature for 15 minutes. To the reaction solution was added tert-butylamine (6.67 ml), and the mixture was stirred at room temperature for 15 minutes. After concentrating the reaction solution under reduced pressure, the residue was azeotroped twice with acetonitrile (5 ml). After adding water (0.240 ml) to a dichloromethane (16.7 ml) solution of the residue, a dichloromethane (16.7 ml) solution of dichloroacetic acid (0.953 ml) was added, and the mixture was stirred at room temperature for 15 minutes. After stopping the reaction by adding pyridine (1.82 ml), the reaction solution was concentrated under reduced pressure. The residue was azeotroped with dehydrated acetonitrile (10 ml) three times, leaving about 5 ml of acetonitrile in the last one. The obtained acetonitrile solution of the title compound was used as it was in the next reaction.

(공정 8) (Process 8)

시판 (ChemGenes) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.31 g) 을 탈수 아세토니트릴 (10 ㎖) 로 3 회 공비하고, 마지막 1 회는 5 ㎖ 정도의 아세토니트릴을 남기고, 몰레큘러 시브스 3A, 1/16 (펠릿상의 것 5 립 (粒)) 을 첨가하였다. 상기 공정 7 에서 합성한 용액에, 상기 서술한 아세토니트릴 용액을 첨가하고, 질소 분위기하, 실온에서 20 분간 교반하였다. 반응액에 N,N-디메틸-N'-(3-술파닐리덴-3H-1,2,4-디티아졸-5-일)메탄이미드아미드 (300 ㎎) 를 첨가하고, 실온에서 30 분간 교반한 후, 반응액을 감압 농축하였다. 잔류물의 디클로로메탄 (19.0 ㎖) 용액에 물 (0.240 ㎖) 을 첨가한 후, 디클로로아세트산 (1.20 ㎖) 의 디클로로메탄 (19.0 ㎖) 용액을 첨가하고, 실온에서 15 분간 교반하였다. 반응액에 피리딘 (13.2 ㎖) 을 첨가하여 반응을 정지시킨 후, 감압 농축하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Commercially available (ChemGenes) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{ (2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.31 g) was azeotroped with dehydrated acetonitrile (10 ml) three times, and the last time was about 5 ml of aceto Leaving the nitrile, Molecular Sieves 3A, 1/16 (5 pellets) were added. The acetonitrile solution described above was added to the solution synthesized in the step 7, and stirred for 20 minutes at room temperature under a nitrogen atmosphere. N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide (300 mg) was added to the reaction solution, and the reaction was carried out at room temperature for 30 minutes. After stirring, the reaction solution was concentrated under reduced pressure. After adding water (0.240 ml) to a solution of the residue in dichloromethane (19.0 ml), a solution of dichloroacetic acid (1.20 ml) in dichloromethane (19.0 ml) was added, and the mixture was stirred at room temperature for 15 minutes. After stopping the reaction by adding pyridine (13.2 ml) to the reaction solution, the mixture was concentrated under reduced pressure. The obtained crude product was used as it was for the next reaction.

(공정 9) (Process 9)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 8 에서 얻어진 미정제 생성물의 피리딘 (39.6 ㎖) 용액을 25 ㎖ 정도까지 농축한 후, 2-클로로-5,5-디메틸-1,3,2λ5-디옥사포스피난-2-온 (908 ㎎) 을 첨가하고, 실온에서 30 분간 교반하였다. 반응액에 물 (0.84 ㎖) 과 3H-1,2-벤조디티올-3-온 (336 ㎎) 을 첨가하고, 실온에서 15 분간 교반하였다. 반응액을 탄산수소나트륨 (5.25 g) 의 수용액 (180 ㎖) 에 따르고, 실온에서 30 분간 교반한 후, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조시킨 후, 건조제를 여과 제거하고 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (507 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. After concentrating the pyridine (39.6 ml) solution of the crude product obtained in step 8 to about 25 ml, 2-chloro-5,5-dimethyl-1,3,2λ 5 -dioxaphosphinan-2-one ( 908 mg) was added and stirred at room temperature for 30 minutes. Water (0.84 ml) and 3H-1,2-benzodithiol-3-one (336 mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was poured into an aqueous solution (180 ml) of sodium hydrogen carbonate (5.25 g), and after stirring at room temperature for 30 minutes, extraction was performed with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to give the title compound (507 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1219(M＋H)＋.MS(ESI)m/z: 1219(M+H) + .

(공정 10) (Process 10)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16- bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (507 ㎎) 의 메탄올 (5 ㎖) 용액에 28 ％ 암모니아수 (5 ㎖) 를 첨가하고, 실온에서 14 시간 교반하였다. 반응액을 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물 (301 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 28% aqueous ammonia (5 ml) was added to a methanol (5 ml) solution of the compound (507 mg) obtained in step 9, and the mixture was stirred at room temperature for 14 hours. After concentrating the reaction solution, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain the title compound (301 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 958(M＋H)＋.MS(ESI) m/z : 958(M+H) + .

(공정 11) (Process 11)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 10 에서 얻어진 화합물 (301 ㎎) 에 트리에틸아민3불화수소산염 (3.84 ㎖) 을 첨가하고, 45 ℃ 에서 3 시간 교반하였다. 실온하, 반응액에 빙랭한 1 M 탄산수소트리에틸암모늄 수용액 (20 ㎖) 과 트리에틸아민 (4 ㎖) 의 혼합액을 첨가하였다. 반응액을 감압 농축 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 25 ％ (0 분 - 40 분)] 로 정제하고, 인 원자 상의 디아스테레오머를 분리하였다. 얻어진 화합물 (트리에틸아민염) 을 이하의 수법으로 나트륨염으로 변환하였다.Triethylamine hydrofluoric acid salt (3.84 ml) was added to the compound (301 mg) obtained in step 10, and the mixture was stirred at 45°C for 3 hours. To the reaction solution at room temperature, an ice-cooled mixture of 1 M aqueous solution of triethylammonium hydrogen carbonate (20 ml) and triethylamine (4 ml) was added. After concentration of the reaction solution under reduced pressure, C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution/acetonitrile, acetonitrile: 0% to 25% (0 min - 40 min)], and the diastereomer on the phosphorus atom was isolated. The obtained compound (triethylamine salt) was converted into a sodium salt by the following method.

[나트륨염으로의 변환] [Conversion to Sodium Salt]

BT AG (등록상표) 50W-X2 Resin (biotechnology grade, 100-200 mesh, hydrogen form) (500 ㎎) 을 순수에 현탁하고, 빈 칼럼에 충전하였다. 과잉의 순수를 자연 유하시킨 후, 1 M 수산화나트륨 수용액 (5 ㎖) 과 순수 (10 ㎖) 를 차례로 자연 유하시켰다. 상기에서 얻어진 화합물을 순수 (5 ㎖) 에 용해하여 칼럼에 차지하였다. 자연 유하한 용액을 분취 후, 추가로 순수 (10 ㎖) 로 용출하였다. 목적물을 포함하는 획분을 아울러 동결 건조시켜, 표제 화합물의 디아스테레오머 1 (83.4 ㎎), 디아스테레오머 2 (44.8 ㎎), 및 디아스테레오머 3 (13.1 ㎎) 을 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2, 3). BT AG (registered trademark) 50W-X2 Resin (biotechnology grade, 100-200 mesh, hydrogen form) (500 mg) was suspended in pure water and charged into an empty column. After the excess pure water was naturally flowed, 1 M sodium hydroxide aqueous solution (5 ml) and pure water (10 ml) were naturally flowed in that order. The compound obtained above was dissolved in pure water (5 ml) and loaded onto a column. After fractionation of the naturally flowing solution, it was further eluted with pure water (10 ml). Fractions containing the target substance were collectively freeze-dried to obtain diastereomer 1 (83.4 mg), diastereomer 2 (44.8 mg), and diastereomer 3 (13.1 mg) of the title compound (retention time by HPLC: stereomer 1 > 2, 3).

디아스테레오머 1 diastereomer 1

Figure pat00123
Figure pat00123

디아스테레오머 2 diastereomer 2

Figure pat00124
Figure pat00124

디아스테레오머 3 diastereomer 3

Figure pat00125
Figure pat00125

실시예 2 : CDN2 의 합성 Example 2: Synthesis of CDN2

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) )-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5, 8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

(실시예 1 에 기재한 화합물 1 의 디아스테레오머 4) (Diastereomer 4 of Compound 1 described in Example 1)

[화학식 110][Formula 110]

Figure pat00126
Figure pat00126

[합성 스킴][Synthesis Scheme]

[화학식 111][Formula 111]

Figure pat00127
Figure pat00127

(공정 1) (Process 1)

N-벤조일-3'-O-[tert-부틸(디메틸)실릴]-2'-O-[하이드록시(옥소)-λ5-포스파닐]아데노신 N-benzoyl-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[hydroxy(oxo)-λ 5 -phosphanyl]adenosine

시판 (ChemGenes) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (962 ㎎) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액을 그대로 다음의 반응에 사용하였다.Commercially available (ChemGenes) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{ The reaction was carried out in the same manner as in Example 1 Step 7 using (2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (962 mg) to obtain acetonitrile of the title compound. got a solution. This acetonitrile solution was used as it was for the next reaction.

(공정 2) (Process 2)

상기 공정 1 에서 얻어진 화합물과 실시예 1 공정 6 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.A reaction was carried out in the same manner as in Example 1 Step 8 using the compound obtained in Step 1 and the compound (1.00 g) obtained in Step 6 of Example 1. The obtained crude product was used as it was for the next reaction.

(공정 3) (Process 3)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2-(2-시아노에톡시)-10-옥소-10-술파닐-2-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-10-oxo-10 -sulfanyl-2-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 2 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (367 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. The reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 2 above to obtain the title compound (367 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1219(M＋H)＋.MS(ESI)m/z: 1219(M+H) + .

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16- bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 3 에서 얻어진 화합물 (367 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (115 ㎎ : 불순물 함유) 과 디아스테레오머 4 (101 ㎎ : 불순물 함유) 를 얻었다. Using the compound (367 mg) obtained in step 3 above, a reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (115 mg: containing impurities) and diastereomer 4 (101 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 958(M＋H)＋. MS(ESI) m/z : 958(M+H) + .

디아스테레오머 4 (고극성) Diastereomer 4 (high polarity)

MS(ESI)m/z : 958(M＋H)＋.MS(ESI) m/z : 958(M+H) + .

(공정 5) (Process 5)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(화합물 1 의 디아스테레오머 4) (Diastereomer 4 of Compound 1)

상기 공정 4 에서 얻어진 화합물 (디아스테레오머 4) (101 ㎎ : 불순물 함유) 를 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (diastereomer 4) (101 mg: containing impurities) obtained in step 4 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by purification under the following [purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 5 ％ - 100 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 5% - 100% (0 min - 40 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (28.5 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Example 1, step 11, to obtain the title compound (28.5 mg).

Figure pat00128
Figure pat00128

실시예 3 : CDN3 의 합성 Example 3: Synthesis of CDN3

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-2,10,15,16-테트라하이드록시-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-14-(6 ,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 112][Formula 112]

Figure pat00129
Figure pat00129

[합성 스킴][Synthesis Scheme]

[화학식 113][Formula 113]

Figure pat00130
Figure pat00130

(공정 1) (Process 1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(oleate)

실시예 1 공정 11 에서 얻어진 화합물 (디아스테레오머 1) (30.0 ㎎) 의 아세톤 (0.5 ㎖) - 물 (0.2 ㎖) 용액에 트리에틸아민 (0.27 ㎖) 과 요오드메탄 (60 ㎕) 을 첨가하여 1 일간 교반하였다. 반응액을 감압 농축 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 20 ％ (0 분 - 40 분)] 로 정제하였다. 얻어진 화합물을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21.2 ㎎) 을 얻었다. Example 1 To a solution of the compound (diastereomer 1) (30.0 mg) obtained in step 11 in acetone (0.5 ml) - water (0.2 ml) was added triethylamine (0.27 ml) and iodomethane (60 µl) to obtain 1 Stirred daily. The reaction solution was concentrated under reduced pressure and then purified by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 0% - 20% (0 min - 40 min)]. The obtained compound was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Example 1 step 11 to obtain the title compound (21.2 mg).

Figure pat00131
Figure pat00131

실시예 4 : CDN4 의 합성Example 4: Synthesis of CDN4

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-di Hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecyne-2,10-dione

[화학식 114][Formula 114]

Figure pat00132
Figure pat00132

[합성 스킴][Synthesis Scheme]

[화학식 115][Formula 115]

Figure pat00133
Figure pat00133

(공정 1) (Process 1)

실시예 1 공정 7 의 반응을 이하의 스케일로 실시하였다 (원료 : 1.01 g). 얻어진 화합물의 아세토니트릴 용액과 시판 (Wuhu Nuowei Chemistry) 되는 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-N-(2-메틸프로파노일)구아노신 (954 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The reaction in step 7 of Example 1 was carried out on the following scale (raw material: 1.01 g). Acetonitrile solution of the obtained compound and commercially available (Wuhu Nuowei Chemistry) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2 Using '-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-N-(2-methylpropanoyl)guanosine (954 mg), Example Reaction was performed by the method similar to 1 process 8. The obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-2-일}-2-메틸프로판아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-methylpropanamide

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (357 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 1 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (357 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1201(M＋H)＋.MS(ESI) m/z : 1201(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H- Purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2, 3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][ 1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 3 에서 얻어진 화합물 (357 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (241 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the compound (357 mg) obtained in step 3 above, a reaction was carried out in the same manner as in step 10 of Example 1 to obtain the title compound (241 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 974(M＋H)＋.MS(ESI) m/z : 974(M+H) + .

(공정 4) (Process 4)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16 -Dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyne-2,10-bis(thiolate)

상기 공정 3 에서 얻어진 화합물 (241 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 인 원자 상의 디아스테레오머를 분리하고, 표제 화합물의 2 종류의 디아스테레오머를 각각 트리에틸아민염으로서 얻었다. Using the compound (241 mg) obtained in step 3 above, reaction was carried out in the same manner as in step 11 of Example 1, and the diastereomer on the phosphorus atom was separated under the following [purification conditions] to obtain two types of the title compound. The diastereomers of were each obtained as triethylamine salts.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 20 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 20% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물의 디아스테레오머 1 (56.7 ㎎) 과 디아스테레오머 2 (25.9 ㎎) 를 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). The obtained triethylamine salt was subjected to salt exchange by the same method as [Conversion to sodium salt] described in Example 1 step 11 to obtain diastereomer 1 (56.7 mg) and diastereomer 2 (25.9 mg) of the title compound. was obtained (retention time of HPLC: diastereomer 1 > 2).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00134
Figure pat00134

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00135
Figure pat00135

실시예 5 : CDN5 의 합성 Example 5: Synthesis of CDN5

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 116][Formula 116]

Figure pat00136
Figure pat00136

[합성 스킴][Synthesis Scheme]

[화학식 117][Formula 117]

Figure pat00137
Figure pat00137

(공정 1) (Process 1)

2',3',5'-트리-O-아세틸-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]이노신 2',3',5'-tri-O-acetyl-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine

2',3',5'-트리-O-아세틸이노신 (5.00 g) 의 테트라하이드로푸란 (90 ㎖) 현탁액에, 2-(트리메틸실릴)에틸(2-하이드록시에틸)카르바메이트 (3.12 g) 와 트리페닐포스핀 (3.99 g) 을 첨가한 후, 디프로판-2-일 (E)-디아젠-1,2-디카르복실레이트 (3.05 ㎖) 의 테트라하이드로푸란 (10 ㎖) 용액을 첨가하고, 실온에서 15 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (3.01 g) 을 얻었다. To a suspension of 2',3',5'-tri-O-acetylinosine (5.00 g) in tetrahydrofuran (90 ml), 2-(trimethylsilyl)ethyl(2-hydroxyethyl)carbamate (3.12 g ) and triphenylphosphine (3.99 g) were added, then a solution of dipropan-2-yl (E) -diazen-1,2-dicarboxylate (3.05 ml) in tetrahydrofuran (10 ml) was added. It was added and stirred at room temperature for 15 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to obtain the title compound (3.01 g).

Figure pat00138
Figure pat00138

(공정 2) (Process 2)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]이노신 5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine

상기 공정 1 에서 얻어진 화합물 (3.01 g) 의 테트라하이드로푸란 (15 ㎖) - 메탄올 (15 ㎖) 용액에 탄산칼륨 (100 ㎎) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에 아세트산 (83 ㎕) 을 첨가하고, 감압 농축하고, 잔류물을 피리딘으로 공비하였다. 재차 피리딘 (30 ㎖) 에 용해 후, 0 ℃ 에서 4,4'-디메톡시트리틸클로라이드 (2.10 g) 를 첨가하고, 30 분간 교반한 후, 냉장고에서 밤새 보관하였다. 반응액에 메탄올 (1㎖) 을 첨가하고, 30 분간 교반한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (3.61 g) 을 얻었다. Potassium carbonate (100 mg) was added to a tetrahydrofuran (15 ml)-methanol (15 ml) solution of the compound (3.01 g) obtained in step 1 above, and the mixture was stirred at room temperature for 2 hours. Acetic acid (83 μl) was added to the reaction solution, and the mixture was concentrated under reduced pressure, and the residue was azeotroped with pyridine. After dissolving in pyridine (30 ml) again, 4,4'-dimethoxytrityl chloride (2.10 g) was added at 0°C, and after stirring for 30 minutes, the mixture was stored in a refrigerator overnight. Methanol (1 ml) was added to the reaction solution, and after stirring for 30 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (3.61 g).

Figure pat00139
Figure pat00139

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 3) (Process 3)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-[2-({[2-(trimethylsilyl) Toxy]carbonyl}amino)ethyl]inosine

상기 공정 2 에서 얻어진 화합물 (3.61 g) 의 디클로로메탄 (18 ㎖) 용액에, 이미다졸 (811 ㎎) 과 tert-부틸(클로로)디메틸실란 (861 ㎎) 을 첨가하고, 실온에서 17 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조시킨 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.61 g) 과 표제 화합물의 위치 이성체인 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]이노신 (1.31 g) 을 각각 얻었다. Imidazole (811 mg) and tert-butyl(chloro)dimethylsilane (861 mg) were added to a solution of the compound (3.61 g) obtained in step 2 above in dichloromethane (18 ml), and the mixture was stirred at room temperature for 17 hours. A saturated aqueous solution of sodium hydrogen carbonate was added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (1.61 g) and its positional isomer 5'-O-[bis(4-methoxyphenyl). )(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine (1.31 g ) were obtained, respectively.

Figure pat00140
Figure pat00140

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00141
Figure pat00141

(공정 4) (Process 4)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine

상기 공정 3 에서 얻어진 화합물 (1.61 g) 의 디클로로메탄 (18.5 ㎖) 용액에, 4,5-디시아노이미다졸 (240 ㎎) 과 2-시아노에틸N,N,N',N'-테트라이소프로필포스포로디아미다이트 (0.703 ㎖) 를 첨가하고, 실온에서 15 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시켰다. 반응액을 디클로로메탄으로 추출 후, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 DIOL 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.95 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 61 : 39) 로서 얻었다.To a dichloromethane (18.5 ml) solution of the compound (1.61 g) obtained in step 3 above, 4,5-dicyanoimidazole (240 mg) and 2-cyanoethyl N, N, N', N'-tetraiso Propylphosphorodiamidite (0.703 ml) was added, and the mixture was stirred at room temperature for 15 hours. The reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution. After extracting the reaction solution with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [hexane/ethyl acetate] to give the title compound (1.95 g) as a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 61 : 39).

Figure pat00142
Figure pat00142

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 910 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (950 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 910 mg). A reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (950 mg) obtained in Step 4 above. The obtained crude product was used as it was for the next reaction.

(공정 6) (Process 6)

2-(트리메틸실릴)에틸 (2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro- 2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoe Toxy)-2-oxo-2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)carbamate

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (602 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 5 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (602 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1303(M＋H)＋.MS(ESI)m/z: 1303(M+H) + .

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-7-{6-옥소-1-[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]-1,6-디하이드로-9H-푸린-9-일}-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2; 10-dioxo-7-{6-oxo-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]-1,6-dihydro-9H-purine-9- yl}-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5 ,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiol rate)

상기 공정 6 에서 얻어진 화합물 (602 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (205 ㎎ : 불순물 함유) 과 디아스테레오머 2 (244 ㎎ : 불순물 함유) 를 얻었다. Using the compound (602 mg) obtained in step 6 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (205 mg: containing impurities) and diastereomer 2 (244 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1146(M＋H)＋. MS(ESI)m/z: 1146(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1146(M＋H)＋.MS(ESI)m/z: 1146(M+H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene- 2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (145 ㎎ : 불순물 함유) 에 트리에틸아민3불화수소산염 (1.31 ㎖) 을 첨가하고, 45 ℃ 에서 3 시간 교반하였다. 실온하, 반응액에 빙랭한 1 M 탄산수소트리에틸암모늄 용액 (10 ㎖) 과 트리에틸아민 (2 ㎖) 의 혼합 용액을 첨가하였다. 반응액을 감압 농축 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하였다. 얻어진 화합물의 테트라하이드로푸란 (4 ㎖) 용액에 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 2 ㎖) 을 첨가하고, 실온에서 39 시간 교반하였다. 반응액에, 10 mM 아세트산트리에틸암모늄 수용액 (4 ㎖) 을 첨가하고, 감압 농축하였다. 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 50 ％ (0 분 - 40 분)] 로 정제하였다. 얻어진 화합물 (트리에틸아민염) 의 염 교환을 실시할 때에, 화합물을 용해하는 용매 및 용출액의 일부에 아세토니트릴-메탄올-순수 (1 : 1 : 1) 의 혼합액을 사용하는 것 이외에는, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (72.5 ㎎) 을 얻었다. Triethylamine hydrofluoric acid salt (1.31 ml) was added to the compound (diastereomer 1) (145 mg: containing impurities) obtained in step 7, and the mixture was stirred at 45°C for 3 hours. To the reaction solution at room temperature, an ice-cooled 1 M triethylammonium hydrogen carbonate solution (10 ml) and a mixed solution of triethylamine (2 ml) were added. The reaction solution was concentrated under reduced pressure and purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile]. A solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 2 ml) was added to a solution of the obtained compound in tetrahydrofuran (4 ml), and the mixture was stirred at room temperature for 39 hours. To the reaction solution, 10 mM aqueous solution of triethylammonium acetate (4 ml) was added, and the mixture was concentrated under reduced pressure. The residue was analyzed by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 50% (0 min - 40 min). )]. Example 1, except that a mixture of acetonitrile-methanol-pure water (1:1:1) was used as a solvent for dissolving the compound and a part of the eluate when performing salt exchange of the obtained compound (triethylamine salt). Salt exchange was carried out by the same method as [Conversion to sodium salt] described in Step 11 to obtain the title compound (72.5 mg).

Figure pat00143
Figure pat00143

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene- 2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (133 ㎎ : 불순물 함유) 을 사용하여 상기 공정 8-1 과 동일한 수법으로 반응 및 염 교환을 실시하고, 표제 화합물 (55.4 ㎎) 을 얻었다. Reaction and salt exchange were carried out in the same manner as in Step 8-1 using the compound (Diastereomer 2) (133 mg: containing impurities) obtained in Step 7 above to obtain the title compound (55.4 mg).

Figure pat00144
Figure pat00144

실시예 6 : CDN6 의 합성 Example 6: Synthesis of CDN6

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro- 9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 118][Formula 118]

Figure pat00145
Figure pat00145

[합성 스킴][Synthesis Scheme]

[화학식 119][Formula 119]

Figure pat00146
Figure pat00146

(공정 1) (Process 1)

2',3',5'-트리-O-아세틸-1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)이노신 2',3',5'-tri-O-acetyl-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine

시판 (Ark Pharm) 되는 2',3',5'-트리-O-아세틸이노신 (10.0 g) 의 테트라하이드로푸란 (100 ㎖) 현탁액에, 2-{[tert-부틸(디메틸)실릴]옥시}에탄-1-올 (5.37 g) 과 트리페닐포스핀 (7.69 g) 을 첨가한 후, 디프로판-2-일 (E)-디아젠-1,2-디카르복실레이트 (6.10 ㎖) 를 첨가하고, 실온에서 6 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/디클로로메탄] 으로 정제하고, 표제 화합물을 트리페닐포스핀옥사이드와의 혼합물 (10.6 g) 로서 얻었다. To a suspension of commercially available (Ark Pharm) 2',3',5'-tri-O-acetylinosine (10.0 g) in tetrahydrofuran (100 ml), 2-{[tert-butyl(dimethyl)silyl]oxy} After adding ethane-1-ol (5.37 g) and triphenylphosphine (7.69 g), dipropan-2-yl (E)-diazen-1,2-dicarboxylate (6.10 mL) was added. and stirred at room temperature for 6 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate/dichloromethane] to obtain the title compound as a mixture with triphenylphosphine oxide (10.6 g).

Figure pat00147
Figure pat00147

(공정 2) (Process 2)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)이노신 5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine

상기 공정 1 에서 얻어진 화합물 (10.6 g) 을 사용하여, 실시예 5 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물을 트리페닐포스핀옥사이드와의 혼합물 (7.21 g) 로서 얻었다. Using the compound (10.6 g) obtained in the above step 1, reaction was carried out in the same manner as in Example 5 step 2 to obtain the title compound as a mixture (7.21 g) with triphenylphosphine oxide.

Figure pat00148
Figure pat00148

(공정 3) (Process 3)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl] oxy} ethyl) inosine

상기 공정 2 에서 얻어진 화합물 (7.21 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.17 g) 과 표제 화합물의 위치 이성체인 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)이노신 (2.55 g) 을 얻었다. Using the compound (7.21 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (2.17 g) and 5'-O-[bis(4), which is a positional isomer of the title compound. -methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine (2.55 g) got

Figure pat00149
Figure pat00149

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00150
Figure pat00150

(공정 4) (Process 4)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl] Oxy}ethyl)-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine

상기 공정 3 에서 얻어진 화합물 (2.17 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.65 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the compound (2.17 g) obtained in step 3 above, a reaction was carried out in the same manner as in step 4 of Example 5 to obtain the title compound (2.65 g) as a diastereomer mixture on a phosphorus atom.

Figure pat00151
Figure pat00151

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 935 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (950 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 935 mg). A reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (950 mg) obtained in Step 4 above. The obtained crude product was used as it was for the next reaction.

(공정 6) (Process 6)

3-({(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-일}옥시)프로판니트릴 3-({(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-{[tert-butyl(dimethyl)silyl] oxy} ethyl) -6-oxo-1,6-dihydro-9H-purin-9-yl] -2-oxo-2-sulfanyl-10-sulfanylidene octahydro-2H, 10H, 12H-5; 8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2,10-yl}oxy) propane nitrile

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (494 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 5 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (494 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1274(M＋H)＋.MS(ESI) m/z : 1274(M+H) + .

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ 5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- [1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano -2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (494 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (88.5 ㎎ : 불순물 함유) 과 디아스테레오머 2 (70.7 ㎎ : 불순물 함유) 를 얻었다. Using the compound (494 mg) obtained in step 6 above, a reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (88.5 mg: containing impurities) and diastereomer 2 (70.7 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1003(M-C6H15Si＋2H)＋. MS(ESI)m/z: 1003(MC 6 H 15 Si + 2H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1003(M-C6H15Si＋2H)＋.MS(ESI)m/z: 1003(MC 6 H 15 Si + 2H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (88.5 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (diastereomer 1) (88.5 mg: containing impurities) obtained in step 7 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by purification under the following [purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (25.7 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (25.7 mg).

Figure pat00152
Figure pat00152

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (70.7 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 2) (70.7 mg: containing impurity) obtained in Step 7 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 15 ％ - 70 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 15% - 70% (0 min - 40 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (17.8 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Example 1 Step 11 to obtain the title compound (17.8 mg).

Figure pat00153
Figure pat00153

실시예 7 : CDN7 의 합성 Example 7: Synthesis of CDN7

N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)-2-하이드록시아세트아미드N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis(sulfanyl) )-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5, 8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo -6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[화학식 120][Formula 120]

Figure pat00154
Figure pat00154

[합성 스킴][Synthesis Scheme]

[화학식 121][Formula 121]

Figure pat00155
Figure pat00155

(공정 1-1) (Process 1-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6-oxo -1,6-dihydro-9H-purin-9-yl} -2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza Benzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

실시예 5 공정 8-1 에서 얻어진 화합물 (10.0 ㎎) 의 N,N-디메틸포름아미드 (0.5 ㎖) 용액에, 트리에틸아민 (8 ㎕) 과 1-[(하이드록시아세틸)옥시]피롤리딘-2,5-디온 (5.3 ㎎) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액을 10 mM 아세트산트리에틸암모늄 수용액으로 희석하고, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 30 ％ (0 분 - 40 분)] 로 정제하였다. 얻어진 화합물 (트리에틸아민염) 을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (10.5 ㎎) 을 얻었다. Example 5 To a solution of the compound (10.0 mg) obtained in step 8-1 in N,N-dimethylformamide (0.5 ml), triethylamine (8 µl) and 1-[(hydroxyacetyl)oxy]pyrrolidine were added. -2,5-dione (5.3 mg) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with an aqueous 10 mM triethylammonium acetate solution, and subjected to C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 0% - 30% (0 min - 40 min)]. The obtained compound (triethylamine salt) was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (10.5 mg).

Figure pat00156
Figure pat00156

(공정 1-2) (Process 1-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6-oxo -1,6-dihydro-9H-purin-9-yl} -2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza Benzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

실시예 5 공정 8-2 에서 얻어진 화합물 (30.0 ㎎) 을 사용하여, 상기 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Example 5 Using the compound (30.0 mg) obtained in Step 8-2, the reaction was carried out in the same manner as in Step 1-1, and then purified under the following [Purification conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)].[Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (23.6 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (23.6 mg).

Figure pat00157
Figure pat00157

실시예 8 : CDN8 의 합성 Example 8: Synthesis of CDN8

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16- Dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

[화학식 122][Formula 122]

Figure pat00158
Figure pat00158

[합성 스킴][Synthesis Scheme]

[화학식 123][Formula 123]

Figure pat00159
Figure pat00159

(공정 1) (Process 1)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2',3'-비스-O-[tert-부틸(디메틸)실릴]-2-클로로아데노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2',3'-bis-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine

이미 알려진 문헌 (J. Med. Chem. 1989, 32, 1135-1140) 의 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-클로로아데노신 (29.1 g) 의 N,N-디메틸포르미아미드 (145 ㎖) 용액에, 이미다졸 (16.4 g) 과 tert-부틸디메틸클로로실란 (18.2 g) 을 첨가하고, 실온에서 18 시간 교반하였다. 반응액에 물을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (34.9 g) 을 얻었다. N of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-chloroadenosine (29.1 g) of known literature (J. Med. Chem. 1989, 32, 1135-1140), To a solution of N-dimethylformiamide (145 ml), imidazole (16.4 g) and tert-butyldimethylchlorosilane (18.2 g) were added, and the mixture was stirred at room temperature for 18 hours. After stopping the reaction by adding water to the reaction solution, extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (34.9 g).

Figure pat00160
Figure pat00160

(공정 2) (Process 2)

N-아세틸-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2',3'-비스-O-[tert-부틸(디메틸)실릴]-2-클로로아데노신 N-acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2',3'-bis-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine

상기 공정 1 에서 얻어진 화합물 (34.9 g) 의 피리딘 (210 ㎖) 용액에, 무수 아세트산 (140 ㎖) 과 4-디메틸아미노피리딘 (515 ㎎) 을 첨가하고, 질소 분위기하, 실온에서 21 시간 교반하였다. 반응액을 디클로로메탄 (100 ㎖) 으로 희석한 후, 포화 탄산수소나트륨 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 디클로로메탄 (210 ㎖) 과 모르폴린 (7.30 ㎖) 을 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (45.4 g : 불순물 함유) 을 얻었다. 얻어진 화합물은 그 이상의 정제를 실시하지 않고 다음의 반응에 사용하였다. Acetic anhydride (140 ml) and 4-dimethylaminopyridine (515 mg) were added to a pyridine (210 ml) solution of the compound (34.9 g) obtained in step 1 above, and the mixture was stirred for 21 hours at room temperature under a nitrogen atmosphere. After diluting the reaction solution with dichloromethane (100 ml), saturated aqueous sodium hydrogen carbonate solution was added, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Dichloromethane (210 ml) and morpholine (7.30 ml) were added to the residue, and the mixture was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (45.4 g: containing impurities). The obtained compound was used for the next reaction without further purification.

Figure pat00161
Figure pat00161

(공정 3) (Process 3)

N-아세틸-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-클로로아데노신 N-acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-chloroadenosine

상기 공정 2 에서 얻어진 화합물 (45.4 g : 불순물 함유) 의 테트라하이드로푸란 (200 ㎖) 용액에, 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1.0 M, 100 ㎖) 을 첨가하고, 질소 분위기하, 실온에서 3 시간 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/아세톤/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (23.0 g) 을 얻었다. To a tetrahydrofuran (200 ml) solution of the compound (45.4 g: containing impurities) obtained in step 2 above, a tetrahydrofuran solution of tetrabutylammonium fluoride (about 1.0 M, 100 ml) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere. was stirred for 3 hours. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/acetone/0.1% triethylamine] to obtain the title compound (23.0 g).

Figure pat00162
Figure pat00162

(공정 4) (Process 4)

N-아세틸-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2-클로로아데노신 N-Acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine

상기 공정 3 에서 얻어진 화합물 (23.0 g) 의 N,N-디메틸포름아미드 (178 ㎖) 용액에, 이미다졸 (5.94 g) 과 tert-부틸디메틸클로로실란 (6.44 g) 을 첨가하고, 질소 분위기하, 실온에서 18 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (9.01 g) 을 얻었다. To a solution of the compound (23.0 g) obtained in step 3 above in N,N-dimethylformamide (178 ml), imidazole (5.94 g) and tert-butyldimethylchlorosilane (6.44 g) were added, and under a nitrogen atmosphere, It was stirred at room temperature for 18 hours. A saturated aqueous solution of sodium hydrogen carbonate was added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (9.01 g).

Figure pat00163
Figure pat00163

(공정 5) (Process 5)

N-아세틸-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2-클로로-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 N-Acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-chloro-2'-O-{( 2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine

상기 공정 4 에서 얻어진 화합물 (9.01 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (10.6 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 65 : 35) 로서 얻었다. Using the compound (9.01 g) obtained in step 4 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (10.6 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 65:35). ) was obtained as

Figure pat00164
Figure pat00164

(공정 6) (Process 6)

N,N-디에틸에탄아미늄 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아세트아미드 2-클로로-9H-푸린-9-일)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-티올레이트 N,N-Diethylethanelaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-acetamide 2-chloro-9H-purin-9-yl)-14-( 6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl( dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 2.06 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 5 에서 얻어진 화합물 (1.98 g) 을 사용하여, 실시예 1 공정 8 및 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (180 ㎎) 과 디아스테레오머 2 (167 ㎎ : 불순물 함유) 를 얻었다. The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 2.06 g). Using the acetonitrile solution of the obtained compound and the compound (1.98 g) obtained in step 5 above, a reaction was carried out in the same manner as in Example 1 step 8 and Example 1 step 9 to obtain diastereomer 1 (180 mg) and diastereomer 2 (167 mg: containing impurities).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00165
Figure pat00165

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1191(M＋H)＋.MS(ESI)m/z: 1191(M+H) + .

(공정 7-1) (Process 7-1)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16- Bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza Benzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (49.6 ㎎) 의 메탄올 (1.28 ㎖) 용액에, 에틸렌디아민 (256 ㎕) 을 첨가하고, 60 ℃ 에서 2 시간 교반한 후, 마이크로 웨이브 반응 장치를 사용하여 120 ℃ 에서 2 시간 반응시켰다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 40 ％ - 70 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 (37.2 ㎎) 을 얻었다.To a methanol (1.28 ml) solution of the compound (diastereomer 1) (49.6 mg) obtained in step 6 above, ethylenediamine (256 µl) was added, and after stirring at 60°C for 2 hours, a microwave reactor was used. and reacted at 120°C for 2 hours. Purification was performed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 40% - 70% (0 min - 30 min)] to obtain the title compound (37.2 mg).

Figure pat00166
Figure pat00166

(공정 7-2) (Process 7-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H -Purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2 ,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (50.0 ㎎ : 불순물 함유) 의 메탄올 (1.29 ㎖) 용액에, 에틸렌디아민 (25.8 ㎕) 을 첨가하고, 60 ℃ 에서 2 시간 교반한 후, 마이크로 웨이브 반응 장치를 사용하여 120 ℃ 에서 2 시간 반응시켰다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 50 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 (23.7 ㎎) 을 얻었다. Ethylenediamine (25.8 µl) was added to a methanol (1.29 ml) solution of the compound (diastereomer 2) (50.0 mg: containing impurities) obtained in step 6, stirred at 60°C for 2 hours, followed by microwave reaction It was made to react at 120 degreeC for 2 hours using the apparatus. Purification was performed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 50% (0 min - 30 min)] to obtain the title compound (23.7 mg).

Figure pat00167
Figure pat00167

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15, 16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7-1 에서 얻어진 화합물 (37.2 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (37.2 mg) obtained in step 7-1 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (16.5 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as described in Example 1 Step 11 [Conversion to sodium salt] to obtain the title compound (16.5 mg).

Figure pat00168
Figure pat00168

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15, 16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7-2 에서 얻어진 화합물 (23.7 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (23.7 mg) obtained in step 7-2 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (14.9 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (14.9 mg).

Figure pat00169
Figure pat00169

실시예 9 : CDN9 의 합성 Example 9: Synthesis of CDN9

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16 -Dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-dione

[화학식 124][Formula 124]

Figure pat00170
Figure pat00170

[합성 스킴][Synthesis Scheme]

[화학식 125][Formula 125]

Figure pat00171
Figure pat00171

(공정 1-1) (Process 1-1)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16 -bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

실시예 8 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (50.1 ㎎) 의 메탄올 (1.29 ㎖) 용액에, 2-아미노에탄올 (258 ㎕) 을 첨가하고, 60 ℃ 에서 2 시간 교반한 후, 마이크로 웨이브 반응 장치를 사용하여 120 ℃ 에서 2 시간 반응시켰다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 용액/아세토니트릴, 아세토니트릴 : 20 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (39.4 ㎎) 을 에탄올아민 유래의 화합물을 포함하는 혼합물로서 얻었다. To a methanol (1.29 ml) solution of the compound (diastereomer 1) (50.1 mg) obtained in Example 8 step 6, 2-aminoethanol (258 µl) was added, and after stirring at 60°C for 2 hours, microwave It was made to react at 120 degreeC for 2 hours using the reaction apparatus. Purified by preparative HPLC [10 mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% - 60% (0 min - 30 min)] to obtain the title compound (39.4 mg) as a mixture containing a compound derived from ethanolamine got as

Figure pat00172
Figure pat00172

(공정 1-2) (Process 1-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]- 9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H- 2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 8 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (49.3 ㎎ : 불순물 함유) 의 메탄올 (1.27 ㎖) 용액에, 2-아미노에탄올 (254 ㎕) 을 첨가하고, 60 ℃ 에서 2 시간 교반한 후, 마이크로 웨이브 반응 장치를 사용하여 120 ℃ 에서 3 시간 반응시켰다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 용액/아세토니트릴, 아세토니트릴 : 20 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 (26.1 ㎎) 을 얻었다. After adding 2-aminoethanol (254 µl) to a methanol (1.27 ml) solution of the compound (diastereomer 2) (49.3 mg: containing impurities) obtained in step 6 of Example 8 and stirring at 60°C for 2 hours, , It was made to react at 120 degreeC for 3 hours using the microwave reactor. Purification by preparative HPLC [10 mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% - 60% (0 min - 30 min)] gave the title compound (26.1 mg).

Figure pat00173
Figure pat00173

(공정 2-1) (Step 2-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15 ,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 1-1 에서 얻어진 혼합물 (39.4 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. After reacting in the same manner as in Example 1 Step 11 using the mixture (39.4 mg) obtained in Step 1-1 above, purification was carried out under the following [Purification conditions] to obtain the title compound as a triethylamine salt. got it

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (16.8 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (16.8 mg).

Figure pat00174
Figure pat00174

(공정 2-2) (Process 2-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15 ,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 1-2 에서 얻어진 화합물 (26.1 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (26.1 mg) obtained in the above step 1-2, the reaction was carried out in the same manner as in Example 1 step 11, and then purified under the following [purification conditions] to obtain the title compound as a triethylamine salt. got it

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (18.6 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (18.6 mg).

Figure pat00175
Figure pat00175

실시예 10 : CDN10 의 합성 Example 10: Synthesis of CDN10

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노-2-메틸프로필)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl)amino]-9H-purin-9-yl}- 15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 126][Formula 126]

Figure pat00176
Figure pat00176

[합성 스킴][Synthesis Scheme]

[화학식 127][Formula 127]

Figure pat00177
Figure pat00177

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노-2-메틸프로필)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl) Amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro -2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 8 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (41.0 ㎎) 의 메탄올 (1.10 ㎖) 용액에, 1,2-디아미노-2-메틸프로판 (210 ㎕) 을 첨가하고, 60 ℃ 에서 2 시간 교반한 후, 마이크로 웨이브 반응 장치를 사용하여 120 ℃ 에서 6 시간 반응시켰다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 용액/아세토니트릴, 아세토니트릴 : 20 ％ - 60 ％ (0 분 - 30 분)] 로 간이 정제하고, 표제 화합물을 (16.3 ㎎ : 불순물 함유) 을 얻었다. 얻어진 화합물은 그 이상의 정제를 실시하지 않고 다음의 반응에 사용하였다. Example 8 To a solution of the compound (diastereomer 1) (41.0 mg) obtained in step 6 in methanol (1.10 ml) was added 1,2-diamino-2-methylpropane (210 µl), and at 60°C 2 After stirring for an hour, it was made to react at 120 degreeC for 6 hours using the microwave reactor. Simple purification by preparative HPLC [10 mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% - 60% (0 min - 30 min)] gave the title compound (16.3 mg: containing impurities). The obtained compound was used for the next reaction without further purification.

MS(ESI)m/z : 1044(M＋H)＋.MS(ESI) m/z : 1044(M+H) + .

(공정 2) (Process 2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노-2-메틸프로필)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl)amino]-9H-purin-9-yl } -15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene- 2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2,10-bis(thiolate)

상기 공정 1 에서 얻어진 화합물 (16.3 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (16.3 mg: containing impurities) obtained in Step 1 above, purifying under the following [Purification conditions], the title compound was obtained as a triethylamine salt. got as

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 30 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (6.4 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (6.4 mg).

Figure pat00178
Figure pat00178

실시예 11 : CDN11 의 합성 Example 11: Synthesis of CDN11

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(아미노메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ 5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy-2 ,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-dione

[화학식 128][Formula 128]

Figure pat00179
Figure pat00179

[합성 스킴][Synthesis Scheme]

[화학식 129][Formula 129]

Figure pat00180
Figure pat00180

(공정 1) (Process 1)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-시아노아데노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-cyanoadenosine

이미 알려진 문헌 (J. Am. Chem. Soc. 1989, 111, 8502-8504) 의 2-시아노아데노신 (440 ㎎) 의 피리딘 (8.00 ㎖) 용액에 4,4'-디메톡시트리틸클로라이드 (642 ㎎) 를 첨가하고, 질소 분위기하, 실온에서 4 시간 교반하였다. 반응액에 메탄올 (10 ㎖) 을 첨가하여 반응을 정지시킨 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (528 ㎎) 을 얻었다. 4,4'-dimethoxytrityl chloride (642 mg) was added, and the mixture was stirred at room temperature in a nitrogen atmosphere for 4 hours. After stopping the reaction by adding methanol (10 ml) to the reaction solution, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (528 mg).

Figure pat00181
Figure pat00181

(공정 2) (Process 2)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]아데노신 5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine

상기 공정 1 에서 얻어진 화합물 (14.3 g) 의 테트라하이드로푸란 (500 ㎖) 용액에 수소화리튬알루미늄의 테트라하이드로푸란 용액 (약 2.5 M, 29.0 ㎖) 을 첨가하고, 질소 분위기하, 40 ℃ 에서 2 시간 교반하였다. 반응액을 빙랭하여 포화 탄산수소나트륨 수용액 (450 ㎖) 을 첨가하고 10 분간 교반한 후, 1-({[2-(트리메틸실릴)에톡시]카르보닐}옥시)피롤리딘-2,5-디온 (25.0 g) 을 첨가하고, 실온에서 18 시간 반응시켰다. 포화 로셸염 수용액을 첨가하고, 2.5 시간 교반한 후, 디클로로메탄/메탄올의 혼합액으로 추출하였다. 유기층을 무수 황산나트륨으로 건조시킨 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (10.8 g) 을 얻었다. A solution of lithium aluminum hydride in tetrahydrofuran (about 2.5 M, 29.0 ml) was added to a solution of the compound (14.3 g) obtained in step 1 in tetrahydrofuran (500 ml), and the mixture was stirred for 2 hours at 40°C under a nitrogen atmosphere. did After ice-cooling the reaction solution, saturated sodium hydrogencarbonate aqueous solution (450 ml) was added and stirred for 10 minutes, followed by 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5- Dione (25.0 g) was added and it was made to react at room temperature for 18 hours. An aqueous saturated Rochelle salt solution was added, and after stirring for 2.5 hours, extraction was performed with a mixture of dichloromethane/methanol. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (10.8 g).

Figure pat00182
Figure pat00182

(공정 3) (Process 3)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine

상기 공정 2 에서 얻어진 화합물 (10.8 g) 의 피리딘 (70.0 ㎖) 용액에 클로로트리메틸실란 (15.0 ㎖) 을 첨가하고, 질소 분위기하, 실온에서 2 시간 교반하였다. 반응액에 염화벤조일 (8.44 ㎖) 을 첨가하고, 추가로 2 시간 교반하였다. 반응액을 0 ℃ 까지 냉각시키고, 물 (21.0 ㎖) 를 첨가하여 10 분간 교반한 후, 28 ％ 암모니아수 (31.4 ㎖) 를 첨가하여 동 온도에서 추가로 20 분간 교반하였다. 실온까지 승온하여 추가로 3 시간 교반한 후, 반응액을 감압 농축하였다. 잔류물을 아세트산에틸에 현탁시키고, 고체를 여과 제거하였다. 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (9.47 g) 을 얻었다. Chlorotrimethylsilane (15.0 ml) was added to a solution of the compound (10.8 g) obtained in step 2 in pyridine (70.0 ml), and the mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. Benzoyl chloride (8.44 ml) was added to the reaction mixture, and the mixture was further stirred for 2 hours. The reaction solution was cooled to 0°C, water (21.0 ml) was added and stirred for 10 minutes, then 28% aqueous ammonia (31.4 ml) was added and stirred for further 20 minutes at the same temperature. After raising the temperature to room temperature and further stirring for 3 hours, the reaction solution was concentrated under reduced pressure. The residue was suspended in ethyl acetate and the solid was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (9.47 g).

Figure pat00183
Figure pat00183

(공정 4) (Process 4)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-[({[2-(trimethylsilyl) )ethoxy]carbonyl}amino)methyl]adenosine

상기 공정 3 에서 얻어진 화합물 (9.47 g) 을 사용하여, 실시예 8 공정 4 와 동일한 수법에 의해 표제 화합물 (3.13 g) 을 얻었다. The title compound (3.13 g) was obtained by the same method as in Example 8 Step 4 using the compound (9.47 g) obtained in Step 3 above.

Figure pat00184
Figure pat00184

(공정 5) (Process 5)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine

상기 공정 4 에서 얻어진 화합물 (1.49 g) 의 디클로로메탄 (15.5 ㎖) 용액에 N,N-디이소프로필에틸아민 (1.58 ㎖) 과 2-시아노에틸N,N-디이소프로필클로로포스포로아미다이트 (1.04 ㎖) 를 첨가하고, 질소 분위기하, 실온에서 2 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 및 C18 실리카 겔 칼럼 크로마토그래피 [아세토니트릴 : 100 ％] 로 정제하고, 표제 화합물 (1.39 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. N, N-diisopropylethylamine (1.58 ml) and 2-cyanoethyl N, N-diisopropylchlorophosphoroamide in dichloromethane (15.5 ml) solution of the compound (1.49 g) obtained in step 4 above (1.04 mL) was added, and the mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] and C18 silica gel column chromatography [acetonitrile: 100%] to obtain the title compound (1.39 g ) was obtained as a mixture of diastereomers on phosphorus atoms (diastereomer ratio = 6:4).

Figure pat00185
Figure pat00185

(공정 6) (Process 6)

N,N-디에틸에탄아미늄 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-벤즈아미드-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]-9H-푸린-9-일}-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-티올레이트 N,N-diethylethanelaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-benzamide-2-[({[2-(trimethylsilyl)ethoxy] carbonyl}amino)methyl]-9H-purin-9-yl}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd ]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2-thiolate

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.94 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 5 에서 얻어진 화합물 (2.19 g) 을 사용하여, 실시예 1 공정 8 및 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (138 ㎎) 과 디아스테레오머 2 (82.8 ㎎) 를 얻었다. The same reaction as in Example 1 Step 7 was carried out on the following scale (raw material: 1.94 g). A reaction was carried out in the same manner as in Example 1 Step 8 and Example 1 Step 9 using the acetonitrile solution of the obtained compound and the compound (2.19 g) obtained in Step 5 above to obtain diastereomer 1 (138 mg) and diastereomer 2 (82.8 mg) were obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00186
Figure pat00186

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00187
Figure pat00187

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 7-1) (Process 7-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[({[2-(trimethylsilyl) Toxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7 ,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (42.3 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (25.9 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 1 Step 10 using the compound (diastereomer 1) (42.3 mg) obtained in Step 6 above to obtain the title compound (25.9 mg).

Figure pat00188
Figure pat00188

(공정 7-2) (Process 7-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[({[2-(trimethylsilyl) Toxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7 ,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (82.8 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (45.2 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 1 Step 10 using the compound (diastereomer 2) (82.8 mg) obtained in Step 6 above to obtain the title compound (45.2 mg).

Figure pat00189
Figure pat00189

(공정 8-1) (Process 8-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[({[2-(trimethylsilyl) Toxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H- 2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 7-1 에서 얻어진 화합물 (25.9 ㎎) 에 트리에틸아민3불화수소산염 (700 ㎕) 을 첨가하고, 55 ℃ 에서 2 시간 교반하였다. 실온하, 반응액에 빙랭한 1 M 탄산트리에틸암모늄 수용액 (3.5 ㎖) 과 트리에틸아민 (1.10 ㎖) 의 혼합액을 첨가하고, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (19.1 ㎎) 을 얻었다. Triethylamine hydrofluoric acid salt (700 µl) was added to the compound (25.9 mg) obtained in step 7-1, and the mixture was stirred at 55°C for 2 hours. To the reaction solution at room temperature, an ice-cooled mixture of 1 M aqueous solution of triethylammonium carbonate (3.5 ml) and triethylamine (1.10 ml) was added, and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 40% (0 min - 30 min)] to obtain the title compound (19.1 mg).

Figure pat00190
Figure pat00190

(공정 8-2) (Process 8-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[({[2-(트리메틸실릴)에톡시]카르보닐}아미노)메틸]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[({[2-(trimethylsilyl) Toxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H- 2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 7-2 에서 얻어진 화합물 (45.2 ㎎) 을 사용하여, 상기 공정 8-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (37.1 ㎎) 을 얻었다. Using the compound (45.2 mg) obtained in step 7-2 above, a reaction was carried out in the same manner as in step 8-1 to obtain the title compound (37.1 mg).

Figure pat00191
Figure pat00191

(공정 9-1) (Process 9-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(아미노메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy -2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H; 10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2; 10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 8-1 에서 얻어진 화합물 (19.1 ㎎) 의 테트라하이드로푸란 (576 ㎕) 용액에, 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 288 ㎕) 을 첨가하고, 질소 분위기하, 실온에서 밤새 교반한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. To a solution of the compound (19.1 mg) obtained in step 8-1 above in tetrahydrofuran (576 µl), a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 288 µl) was added, and the mixture was stirred at room temperature in a nitrogen atmosphere. After stirring overnight, purification was carried out under the following [Purification conditions] to obtain the title compound as a triethylamine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 30 ％ (0 분 - 40 분)] 및 Sep-Pak (등록상표) C18 [물/아세토니트릴]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 0% - 30% (0 min - 40 min)] and Sep-Pak (registered trademark) C18 [water/acetonitrile].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (8.4 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (8.4 mg).

Figure pat00192
Figure pat00192

(공정 9-2) (Process 9-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(아미노메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy -2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H; 10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2; 10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 8-2 에서 얻어진 화합물 (37.1 ㎎) 을 사용하여, 상기 공정 9-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (12.8 ㎎) 을 얻었다. A reaction was carried out in the same manner as in Step 9-1 using the compound (37.1 mg) obtained in Step 8-2 to obtain the title compound (12.8 mg).

Figure pat00193
Figure pat00193

실시예 12 : CDN12 의 합성 Example 12: Synthesis of CDN12

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(하이드록시메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydroxy- 2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-dione

[화학식 130][Formula 130]

Figure pat00194
Figure pat00194

[합성 스킴][Synthesis Scheme]

[화학식 131][Formula 131]

Figure pat00195
Figure pat00195

(공정 1) (Process 1)

6-클로로-2-요오드-9-{2,3,5-트리스-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 6-Chloro-2-iodo-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine

시판 (Amadis Chemical) 되는 6-클로로-2-요오드-9-β-D-리보퓨라노실-9H-푸린 (9.65 g) 의 에틸렌글리콜디메틸에테르 (120 ㎖) 용액에, 0 ℃ 에서 N,N-디이소프로필에틸아민 (40.7 ㎖) 과 tert-부틸디메틸실릴트리플루오로메탄술포네이트 (26.9 ㎖) 를 첨가하고, 질소 분위기하, 실온까지 승온하여 19 시간 교반하였다. 반응액을 0 ℃ 까지 냉각시키고, 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (13.7 g) 을 얻었다. To a solution of commercially available (Amadis Chemical) 6-chloro-2-iodine-9-β-D-ribofuranosyl-9H-purine (9.65 g) in ethylene glycol dimethyl ether (120 ml), N, N at 0 ° C. -Diisopropylethylamine (40.7 ml) and tert-butyldimethylsilyltrifluoromethanesulfonate (26.9 ml) were added, and the mixture was heated to room temperature under a nitrogen atmosphere and stirred for 19 hours. The reaction solution was cooled to 0°C, and after stopping the reaction by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (13.7 g).

Figure pat00196
Figure pat00196

(공정 2) (Process 2)

2-[(벤조일옥시)메틸]-6-클로로-9-{2,3,5-트리스-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 2-[(benzoyloxy)methyl]-6-chloro-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine

상기 공정 1 에서 얻어진 화합물 (13.7 g) 의 테트라하이드로푸란 (121 ㎖) 용액에, 질소 분위기하에서 테트라키스(트리페닐포스핀)팔라듐(0) (2.10 g) 과 하기의 수법으로 조제한 벤질옥시메틸요오드화아연 (약 0.9 M, 30.2 ㎖) 을 첨가하고, 실온에서 20 시간 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (7.29 g) 을 얻었다. Benzyloxymethyl iodide prepared by the following method with tetrakis(triphenylphosphine)palladium(0) (2.10 g) in a tetrahydrofuran (121 ml) solution of the compound (13.7 g) obtained in the above step 1 under a nitrogen atmosphere. Zinc (about 0.9 M, 30.2 ml) was added and stirred at room temperature for 20 hours. After stopping the reaction by adding a saturated aqueous solution of ammonium chloride to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (7.29 g).

[벤질옥시메틸요오드화아연의 조제] [Preparation of benzyloxymethyl zinc iodide]

질소 분위기하, 아연 분말 (5.99 g) 의 테트라하이드로푸란 (17.1 ㎖) 현탁액을 초음파 처리한 후, 요오드메틸벤조에이트 (12.0 g) 의 테트라하이드로푸란 (21.3 ㎖) 용액을 10 ∼ 15 ℃ 에서 첨가하고, 동 온도에서 1.5 시간 교반하고, 벤질옥시메틸요오드화아연의 테트라하이드로푸란 용액 (약 0.9 M, 38.4 ㎖) 을 얻었다. A suspension of zinc powder (5.99 g) in tetrahydrofuran (17.1 ml) was sonicated under a nitrogen atmosphere, and then a solution of iodomethyl benzoate (12.0 g) in tetrahydrofuran (21.3 ml) was added at 10 to 15°C. , and stirred at the same temperature for 1.5 hours to obtain a solution of benzyloxymethyl zinc iodide in tetrahydrofuran (about 0.9 M, 38.4 ml).

Figure pat00197
Figure pat00197

(공정 3) (Process 3)

2-[(벤조일옥시)메틸]-6-클로로-9-β-D-리보퓨라노실-9H-푸린 2-[(benzoyloxy)methyl]-6-chloro-9-β-D-ribofuranosyl-9H-purine

상기 공정 2 에서 얻어진 화합물 (7.29 g) 의 테트라하이드로푸란 (47.7 ㎖) 용액에, 질소 분위기, 0 ℃ 에서 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 38 ㎖) 을 첨가하고, 동 온도에서 2.5 시간 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (3.69 g) 을 얻었다. To a tetrahydrofuran (47.7 ml) solution of the compound (7.29 g) obtained in step 2 above, a tetrahydrofuran solution of tetrabutylammonium fluoride (about 1 M, 38 ml) was added at 0°C in a nitrogen atmosphere, and the mixture was heated at the same temperature. was stirred for 2.5 hours. After stopping the reaction by adding a saturated aqueous solution of ammonium chloride to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (3.69 g).

Figure pat00198
Figure pat00198

(공정 4) (Process 4)

2-[(벤조일옥시)메틸]-9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-리보퓨라노실}-6-클로로-9H-푸린 2-[(benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl}-6-chloro-9H-purine

상기 공정 3 에서 얻어진 화합물 (2.36 g) 의 피리딘 (56 ㎖) 용액에, 4,4'-디메톡시트리틸클로라이드 (2.30 g) 를 첨가하고, 질소 분위기하, 실온에서 17 시간 교반하였다. 반응액에 에탄올 (20 ㎖) 을 첨가하여, 추가로 10 분간 정도 교반한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.41 g) 을 얻었다. 4,4'-dimethoxytrityl chloride (2.30 g) was added to a pyridine (56 ml) solution of the compound (2.36 g) obtained in the above step 3, and the mixture was stirred at room temperature under a nitrogen atmosphere for 17 hours. Ethanol (20 ml) was added to the reaction solution, and after stirring for about 10 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.41 g).

Figure pat00199
Figure pat00199

(공정 5) (Process 5)

2-[(벤조일옥시)메틸]-9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6-클로로-9H-푸린 2-[(benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D- Ribofuranosyl} -6-chloro-9H-purine

상기 공정 4 에서 얻어진 화합물 (2.61 g) 의 에틸렌글리콜디메틸에테르 (72.0 ㎖) 용액에, N,N-디이소프로필에틸아민 (1.89 ㎖) 과 tert-부틸디메틸실릴트리플루오로메탄술포네이트 (1.24 ㎖) 를 첨가하고, 질소 분위기하, 실온에서 1.5 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.10 g) 을 얻었다. To a solution of the compound (2.61 g) obtained in step 4 in ethylene glycol dimethyl ether (72.0 ml), N, N-diisopropylethylamine (1.89 ml) and tert-butyldimethylsilyltrifluoromethanesulfonate (1.24 ml) ) was added, and the mixture was stirred at room temperature in a nitrogen atmosphere for 1.5 hours. After stopping the reaction by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (1.10 g).

Figure pat00200
Figure pat00200

(공정 6) (Process 6)

2-[(벤조일옥시)메틸]-9-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-2-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-6-클로로-9H-푸린 2-[(benzoyloxy)methyl]-9-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O- {(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6-chloro-9H-purine

상기 공정 5 에서 얻어진 화합물 (511 ㎎) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (569 ㎎) 을 인 원자에 관한 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. Using the compound (511 mg) obtained in step 5 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (569 mg) was obtained with a mixture of diastereomers (diastereomer ratio = 6: 4) was obtained as

Figure pat00201
Figure pat00201

(공정 7) (Process 7)

N,N-디에틸에탄아미늄 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-[(벤조일옥시)메틸]-6-클로로-9H-푸린-9-일}-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-티올레이트 N,N-diethylethanelaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-[(benzoyloxy)methyl]-6-chloro-9H-purine-9- yl}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis {[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 2.72 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 6 에서 얻어진 화합물 (3.03 g) 을 사용하여, 실시예 1 공정 8 및 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (351 ㎎) 과 디아스테레오머 2 (351 ㎎) 를 얻었다. The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 2.72 g). A reaction was carried out in the same manner as in Example 1 Step 8 and Example 1 Step 9 using the acetonitrile solution of the obtained compound and the compound (3.03 g) obtained in Step 6 above to obtain diastereomer 1 (351 mg) and diastereomer 2 (351 mg).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00202
Figure pat00202

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00203
Figure pat00203

(공정 8-1) (Process 8-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-아미노-2-(하이드록시메틸)-9H-푸린-9-일]-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purine-9 -yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5 ,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3 ,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (37.3 ㎎) 의 메탄올 (0.500 ㎖) 용액에, 28 ％ 암모니아 수용액 (0.500 ㎖) 을 첨가하고, 실드 튜브 중 60 ℃ 에서 3 시간 교반하였다. 반응액을 그대로 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 35 ％ - 55 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (22.0 ㎎ : 불순물 함유) 을 얻었다. To a methanol (0.500 ml) solution of the compound (diastereomer 1) (37.3 mg) obtained in step 7 above, 28% ammonia aqueous solution (0.500 ml) was added, and the mixture was stirred at 60°C for 3 hours in a sealed tube. The reaction solution was purified as it was by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 35% - 55% (0 min - 30 min)] to obtain the title compound (22.0 mg: contains impurities).

MS(ESI)m/z : 988(M＋H)＋.MS(ESI) m/z : 988(M+H) + .

(공정 8-2) (Process 8-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-아미노-2-(하이드록시메틸)-9H-푸린-9-일]-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purine-9 -yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5 ,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3 ,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (37.7 ㎎) 의 테트라하이드로푸란 (0.500 ㎖) 용액에, 28 ％ 암모니아 수용액 (0.500 ㎖) 을 첨가하고, 실드 튜브 중 60 ℃ 에서 3 시간 교반하였다. 28 ％ 암모니아 수용액 (0.500 ㎖) 을 추가하고 3 시간 더 교반하였다. 28 ％ 암모니아 수용액 (0.500 ㎖) 을 추가하고 밤새 더 교반하였다. 반응액을 그대로 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 50 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (19.3 ㎎) 을 얻었다. A 28% aqueous ammonia solution (0.500 ml) was added to a solution of the compound (diastereomer 2) (37.7 mg) obtained in step 7 in tetrahydrofuran (0.500 ml), and the mixture was stirred at 60°C for 3 hours in a shielded tube. A 28% aqueous ammonia solution (0.500 ml) was added and stirred for further 3 hours. A 28% aqueous ammonia solution (0.500 mL) was added and further stirred overnight. The reaction mixture was purified as it was by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 20% - 50% (0 min - 30 min)] to obtain the title compound (19.3 mg).

Figure pat00204
Figure pat00204

(공정 9-1) (Process 9-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(하이드록시메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydro Roxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

상기 공정 8-1 에서 얻어진 화합물 (22.0 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (22.0 mg: containing impurity) obtained in the above step 8-1, the reaction was carried out in the same manner as in Example 1 step 11, and then purified under the following [purification conditions]. The title compound was obtained by triethyl It was obtained as an amine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (8.0 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (8.0 mg).

Figure pat00205
Figure pat00205

(공정 9-2) (Process 9-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-(하이드록시메틸)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydro Roxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

상기 공정 8-2 에서 얻어진 화합물 (19.3 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (19.3 mg) obtained in the above step 8-2, the reaction was carried out in the same manner as in Example 1 step 11, and then purified under the following [purification conditions] to obtain the title compound as a triethylamine salt. got it

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (6.5 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Example 1 step 11 to obtain the title compound (6.5 mg).

Figure pat00206
Figure pat00206

실시예 13 : CDN13 의 합성 Example 13: Synthesis of CDN13

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy- 2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-dione

[화학식 132][Formula 132]

Figure pat00207
Figure pat00207

[합성 스킴][Synthesis Scheme]

[화학식 133][Formula 133]

Figure pat00208
Figure pat00208

(공정 1) (Process 1)

9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6-클로로-9H-푸린 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6-chloro- 9H-purine

이미 알려진 문헌 (J. Org. Chem. 2000, 65, 5104-5113) 의 9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-리보퓨라노실}-6-클로로-9H-푸린 (15.3 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (8.44 g) 과 표제 화합물의 위치 이성체인 9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6-클로로-9H-푸린 (5.77 g) 을 얻었다. 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl} of known literature (J. Org. Chem. 2000, 65, 5104-5113) Using -6-chloro-9H-purine (15.3 g), the reaction was carried out in the same manner as in Example 5 Step 3 to obtain the title compound (8.44 g) and 9-{5-O-, which is a positional isomer of the title compound. [bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6-chloro-9H-purine (5.77 g) got

Figure pat00209
Figure pat00209

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00210
Figure pat00210

(공정 2) (Process 2)

9-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-2-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-6-클로로-9H-푸린 9-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6-chloro-9H-purine

상기 공정 1 에서 얻어진 화합물 (5.39 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.78 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (5.39 g) obtained in step 1 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (5.78 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00211
Figure pat00211

(공정 3) (Process 3)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.84 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 2 에서 얻어진 화합물 (1.62 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 1.84 g). The reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (1.62 g) obtained in the above Step 2. The obtained crude product was used as it was for the next reaction.

(공정 4) (Process 4)

3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-(6-클로로-9H-푸린-9-일)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-일]옥시}프로판니트릴 3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- Tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-(6-chloro-9H-purin-9-yl)-2- Oxo-2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano- 2λ 5,10λ-furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecin-10-yl]oxy}propanenitrile

상기 공정 3 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (626 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 3 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (626 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1134(M＋H)＋.MS(ESI)m/z: 1134(M+H) + .

(공정 5) (Process 5)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-Diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-[(2-aminoethyl)amino]-9H-purine-9 -yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5 ,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3 ,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 4 에서 얻어진 화합물 (299 ㎎ : 디아스테레오머 혼합물) 의 에탄올 (10 ㎖) 용액에 에틸렌디아민 (0.352 ㎖) 과 트리에틸아민 (0.735 ㎖) 을 첨가하고, 60 ℃ 에서 15 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (122 ㎎ : 불순물 함유) 과 디아스테레오머 2 (111 ㎎ : 불순물 함유) 를 얻었다. Ethylenediamine (0.352 ml) and triethylamine (0.735 ml) were added to an ethanol (10 ml) solution of the compound (299 mg: diastereomer mixture) obtained in step 4, and the mixture was stirred at 60°C for 15 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (122 mg: containing impurities) and diastereomer of the title compound. 2 (111 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1001(M＋H)＋. MS(ESI) m/z : 1001(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1001(M＋H)＋.MS(ESI) m/z : 1001(M+H) + .

(공정 6-1) (Step 6-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydride Roxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 5 에서 얻어진 화합물 (디아스테레오머 1) (122 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (diastereomer 1) (122 mg: containing impurities) obtained in step 5 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by purification under the following [purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (29.6 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (29.6 mg).

Figure pat00212
Figure pat00212

(공정 6-2) (Step 6-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydride Roxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 5 에서 얻어진 화합물 (디아스테레오머 2) (119 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 2) (119 mg: containing impurity) obtained in Step 5 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (15.6 ㎎) 을 얻었다. The resulting triethylamine salt was subjected to salt exchange in the same manner as described in Example 1, step 11 [Conversion to sodium salt] to obtain the title compound (15.6 mg).

Figure pat00213
Figure pat00213

실시예 14 : CDN14 의 합성 Example 14: Synthesis of CDN14

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-{6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purin-9-yl} -2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro -2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -2,10-dione

[화학식 134][Formula 134]

Figure pat00214
Figure pat00214

[합성 스킴][Synthesis Scheme]

[화학식 135][Formula 135]

Figure pat00215
Figure pat00215

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-{6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- {6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3, 5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 13 공정 4 에서 얻어진 화합물 (313 ㎎) 의 에탄올 (10 ㎖) 용액에, 2-아미노에탄올 (0.330 ㎖) 과 트리에틸아민 (0.769 ㎖) 을 첨가하고, 60 ℃ 에서 15 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (111 ㎎ : 불순물 함유) 과 디아스테레오머 2 (102 ㎎ : 불순물 함유) 를 얻었다. Example 13 To a solution of the compound (313 mg) obtained in step 4 in ethanol (10 ml), 2-aminoethanol (0.330 ml) and triethylamine (0.769 ml) were added, and the mixture was stirred at 60°C for 15 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (111 mg: containing impurities) and diastereomer of the title compound. 2 (102 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1002(M＋H)＋. MS(ESI) m/z : 1002(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1002(M＋H)＋.MS(ESI) m/z : 1002(M+H) + .

(공정 2-1) (Step 2-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-{6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purine-9- yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 1) (111 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (diastereomer 1) (111 mg: containing impurities) obtained in step 1 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by purification under the following [purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (47.1 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (47.1 mg).

Figure pat00216
Figure pat00216

(공정 2-2) (Process 2-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-{6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purine-9- yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 2) (102 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 2) (102 mg: containing impurity) obtained in Step 1 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (27.1 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (27.1 mg).

Figure pat00217
Figure pat00217

실시예 15 : CDN15 의 합성 Example 15: Synthesis of CDN15

N-[2-({9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}아미노)에틸]-2-하이드록시아세트아미드N-[2-({9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis(alcohol) Fanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5 ,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H- Purin-6-yl}amino)ethyl]-2-hydroxyacetamide

[화학식 136][Formula 136]

Figure pat00218
Figure pat00218

[합성 스킴][Synthesis Scheme]

[화학식 137][Formula 137]

Figure pat00219
Figure pat00219

(공정 1) (Process 1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-(6-{[2-(2-하이드록시아세트아미드)에틸]아미노}-9H-푸린-9-일)-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-(6-{[2-(2-hydroxyacetamide)ethyl]amino}- 9H-purin-9-yl)-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2 -yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa Diphosphacyclotetradecine-2,10-bis(thiolate)

실시예 13 공정 6-2 에서 얻어진 화합물 (10.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (10.0 mg) obtained in Example 13 step 6-2, the reaction was carried out in the same manner as in Example 7 step 1-1, and then purified under the following [purification conditions] to obtain the title compound Obtained as ethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 35 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 35% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (6.6 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (6.6 mg).

Figure pat00220
Figure pat00220

실시예 16 : CDN16 의 합성 Example 16: Synthesis of CDN16

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-아미노-6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16 -Dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-dione

[화학식 138][Formula 138]

Figure pat00221
Figure pat00221

[합성 스킴][Synthesis Scheme]

[화학식 139][Formula 139]

Figure pat00222
Figure pat00222

(공정 1) (Process 1)

2-아세트아미드-2',3',5'-트리-O-아세틸-N-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아데노신 2-acetamide-2',3',5'-tri-O-acetyl-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine

시판 (도쿄 화성 공업) 되는 6-클로로-9-β-D-리보퓨라노실-9H-푸린-2-아민 (5.00 g) 의 에탄올 (30 ㎖) 용액에 2-{[tert-부틸(디메틸)실릴]옥시}에탄-1-아민 (3.49 g) 과 N,N-디이소프로필에틸아민 (4.33 ㎖) 을 첨가하고, 80 ℃ 에서 65 시간 교반하였다. 반응액을 감압 농축 후, 잔류물에 피리딘 (15 ㎖) 과 무수 아세트산 (15 ㎖) 을 첨가하고, 70 ℃ 에서 4 시간 교반하였다. 반응액을 감압 농축 후, 포화 탄산수소나트륨 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (8.91 g) 을 얻었다. 2-{[tert-butyl (dimethyl )silyl]oxy}ethane-1-amine (3.49 g) and N,N-diisopropylethylamine (4.33 ml) were added, and the mixture was stirred at 80°C for 65 hours. After concentrating the reaction solution under reduced pressure, pyridine (15 ml) and acetic anhydride (15 ml) were added to the residue, and the mixture was stirred at 70°C for 4 hours. After concentrating the reaction solution under reduced pressure, a saturated aqueous solution of sodium hydrogen carbonate was added, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (8.91 g).

Figure pat00223
Figure pat00223

(공정 2) (Process 2)

2-아세트아미드-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-N-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아데노신 2-acetamide-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine

상기 공정 1 에서 얻어진 화합물 (4.00 g) 의 디클로로메탄 (40 ㎖) 용액에 나트륨메톡시드의 메탄올 용액 (1.0 M, 6.64 ㎖) 을 첨가하고, 0 ℃ 에서 1 시간 교반하였다. 반응액에 아세트산 (0.413 ㎖) 과 피리딘 (0.5 ㎖) 을 첨가하여 반응을 정지시킨 후, 반응액을 감압 농축하였다. 잔류물에 피리딘을 첨가한 후, 감압하에서 일부 농축하고, 피리딘 (약 20 ㎖) 용액을 조제하였다. 이 용액에 0 ℃ 에서 4,4'-디메톡시트리틸클로라이드 (4.68 g) 를 첨가하고, 동 온도에서 30 분간 교반한 후, 4 ℃ 에서 밤새 보존하였다. 반응액에 메탄올 (2 ㎖) 을 첨가하고, 30 분간 교반한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (4.41 g) 을 얻었다. A methanol solution of sodium methoxide (1.0 M, 6.64 ml) was added to a dichloromethane (40 ml) solution of the compound (4.00 g) obtained in step 1 above, and the mixture was stirred at 0°C for 1 hour. After stopping the reaction by adding acetic acid (0.413 ml) and pyridine (0.5 ml) to the reaction solution, the reaction solution was concentrated under reduced pressure. After adding pyridine to the residue, it was partially concentrated under reduced pressure to prepare a pyridine (about 20 ml) solution. To this solution, 4,4'-dimethoxytrityl chloride (4.68 g) was added at 0°C, stirred at the same temperature for 30 minutes, and stored at 4°C overnight. Methanol (2 ml) was added to the reaction solution, and after stirring for 30 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (4.41 g).

Figure pat00224
Figure pat00224

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 3) (Process 3)

2-아세트아미드-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-N-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아데노신 2-acetamide-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl (dimethyl)silyl]oxy}ethyl)adenosine

상기 공정 2 에서 얻어진 화합물 (4.41 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.75 g) 및 표제 화합물의 위치 이성체인 2-아세트아미드-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-N-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아데노신 (1.31 g) 을 얻었다. Using the compound (4.41 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (1.75 g) and 2-acetamide-5'-O, which is a positional isomer of the title compound. -[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl) Adenosine (1.31 g) was obtained.

Figure pat00225
Figure pat00225

(관측 가능한 피크만을 기재) (list only observable peaks)

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00226
Figure pat00226

(공정 4) (Process 4)

2-아세트아미드-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-N-(2-{[tert-부틸(디메틸)실릴]옥시}에틸)-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 2-acetamide-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl (dimethyl)silyl]oxy}ethyl)-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine

상기 공정 3 에서 얻어진 화합물 (1.75 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.08 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. Using the compound (1.75 g) obtained in step 3 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (2.08 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 6:4). ) was obtained as

Figure pat00227
Figure pat00227

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 981 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (1.05 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 981 mg). A reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (1.05 g) obtained in Step 4 above. The obtained crude product was used as it was for the next reaction.

(공정 6) (Process 6)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-[(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아미노]-9H-푸린-2-일}아세트아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-[(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)amino]-9H-purin-2-yl}acetamide

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (413 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. A reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 5 above to obtain the title compound (413 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1330(M＋H)＋.MS(ESI) m/z : 1330(M+H) + .

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-아미노-6-[(2-{[tert-부틸(디메틸)실릴]옥시}에틸)아미노]-9H-푸린-9-일}-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-amino-6-[(2-{[tert-butyl(dimethyl )silyl]oxy}ethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7 ,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (413 ㎎) 의 메탄올 (5 ㎖) 과 28 ％ 암모니아수 (5 ㎖) 에 용해시키고, 실온에서 63 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 간이 정제하고, 저극성의 디아스테레오머 1 과 고극성의 디아스테레오머 2 를 분리하였다. 각각의 디아스테레오머를 재차 메탄올 (5 ㎖) 과 28 ％ 암모니아수 (5 ㎖) 에 용해시키고, 100 ℃ 에서 2 일간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (70.4 ㎎ : 불순물 함유) 과 디아스테레오머 2 (65.1 ㎎ : 불순물 함유) 를 얻었다. The compound (413 mg) obtained in the above step 6 was dissolved in methanol (5 ml) and 28% aqueous ammonia (5 ml), and stirred at room temperature for 63 hours. After concentration of the reaction solution under reduced pressure, the residue was simply purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to separate low-polarity diastereomer 1 and high-polarity diastereomer 2. did Each diastereomer was again dissolved in methanol (5 ml) and 28% aqueous ammonia (5 ml), and stirred at 100°C for 2 days. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (70.4 mg: containing impurities) and diastereomer of the title compound. 2 (65.1 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1131(M＋H)＋. MS(ESI)m/z: 1131(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1131(M＋H)＋.MS(ESI)m/z: 1131(M+H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-아미노-6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15 ,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (70.4 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 1) (70.4 mg: containing impurity) obtained in Step 7 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (20.8 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Example 1 step 11 to obtain the title compound (20.8 mg).

Figure pat00228
Figure pat00228

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-아미노-6-[(2-하이드록시에틸)아미노]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15 ,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (65.1 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 2) (65.1 mg: containing impurities) obtained in Step 7 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 0 ％ - 60 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 0% - 60% (0 min - 40 min)].

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (7.9 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (7.9 mg).

Figure pat00229
Figure pat00229

실시예 17 : CDN17 의 합성 Example 17: Synthesis of CDN17

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[6-(하이드록시메틸)-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[6-(hydroxymethyl)-9H-purin-9-yl]-2,10- bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H; 12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10- Dion

[화학식 140][Formula 140]

Figure pat00230
Figure pat00230

[합성 스킴][Synthesis Scheme]

[화학식 141][Formula 141]

Figure pat00231
Figure pat00231

(공정 1) (Process 1)

6-[(벤조일옥시)메틸]-9-{2,3,5-트리스-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 6-[(benzoyloxy)methyl]-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine

이미 알려진 문헌 (J. Org. Chem. 1997, 62, 6833-6841) 의 6-클로로-9-{2,3,5-트리스-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 (10.7 g) 을 사용하여, 실시예 12 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (10.4 g) 을 얻었다. 6-Chloro-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D- of the known literature (J. Org. Chem. 1997, 62, 6833-6841) The reaction was carried out in the same manner as in Example 12 Step 2 using ribofuranosyl} -9H-purine (10.7 g) to obtain the title compound (10.4 g).

Figure pat00232
Figure pat00232

(공정 2) (Process 2)

6-[(벤조일옥시)메틸]-9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-리보퓨라노실}-9H-푸린 6-[(benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl}-9H-purine

상기 공정 1 에서 얻어진 화합물 (10.3 g) 의 테트라하이드로푸란 (50 ㎖) 용액에, 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 49.4 ㎖) 을 첨가하고, 0 ℃ 에서 4 시간 교반하였다. 반응액에 아세트산 (2.83 ㎖) 을 첨가한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 조 (粗) 정제하였다. 미정제 생성물의 피리딘 (50 ㎖) 용액에, 4,4'-디메톡시트리틸클로라이드 (10.1 g) 를 0 ℃ 에서 첨가하고, 4 ℃ 에서 밤새 교반하였다. 반응액에 메탄올 (2 ㎖) 을 첨가하고, 1 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (4.62 g) 을 얻었다. To a tetrahydrofuran (50 ml) solution of the compound (10.3 g) obtained in step 1 above, a tetrahydrofuran solution of tetrabutylammonium fluoride (about 1 M, 49.4 ml) was added, and the mixture was stirred at 0°C for 4 hours. After adding acetic acid (2.83 ml) to the reaction mixture, the mixture was concentrated under reduced pressure. The residue was crudely purified by silica gel column chromatography [dichloromethane/methanol]. To a solution of the crude product in pyridine (50 ml), 4,4'-dimethoxytritylchloride (10.1 g) was added at 0°C, and the mixture was stirred at 4°C overnight. Methanol (2 ml) was added to the reaction solution, and the mixture was stirred for 1 hour. A saturated aqueous solution of sodium hydrogen carbonate was added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (4.62 g).

Figure pat00233
Figure pat00233

(공정 3) (Process 3)

6-[(벤조일옥시)메틸]-9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 6-[(benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D- Ribofuranosyl} -9H-purine

상기 공정 2 에서 얻어진 화합물 (4.53 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.09 g) 및 표제 화합물의 위치 이성체인 6-[(벤조일옥시)메틸]-9-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-9H-푸린 (1.81 g) 을 얻었다. Using the compound (4.53 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (2.09 g) and the positional isomer of the title compound, 6-[(benzoyloxy)methyl] -9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine (1.81 g) was obtained.

Figure pat00234
Figure pat00234

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00235
Figure pat00235

(공정 4) (Process 4)

6-[(벤조일옥시)메틸]-9-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-2-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-9H-푸린 6-[(benzoyloxy)methyl]-9-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O- {(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-9H-purine

상기 공정 3 에서 얻어진 화합물 (2.04 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.37 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 56 : 44) 로서 얻었다. Using the compound (2.04 g) obtained in step 3 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (2.37 g) was obtained with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 56:44). ) was obtained as

Figure pat00236
Figure pat00236

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 783 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (765 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 783 mg). A reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (765 mg) obtained in Step 4 above. The obtained crude product was used as it was for the next reaction.

(공정 6) (Process 6)

{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}메틸 벤조에이트 {9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-alcohol Panyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}methyl benzoate

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (345 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. A reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 5 above to obtain the title compound (345 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1234(M＋H)＋.MS(ESI) m/z : 1234(M+H) + .

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[6-(하이드록시메틸)-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- [6-(hydroxymethyl)-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (345 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (101 ㎎ : 불순물 함유) 과 디아스테레오머 2 (89.9 ㎎ : 불순물 함유) 를 얻었다. Using the compound (345 mg) obtained in step 6 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (101 mg: containing impurities) and diastereomer 2 (89.9 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 973(M＋H)＋. MS(ESI) m/z : 973(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 973(M＋H)＋.MS(ESI) m/z : 973(M+H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[6-(하이드록시메틸)-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[6-(hydroxymethyl)-9H-purin-9-yl]-2, 10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H -5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis (thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (101 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (Diastereomer 1) (101 mg: containing impurities) obtained in Step 7 above, reaction was carried out in the same manner as in Step 11 of Example 1, followed by purification under the following [Purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (59.2 ㎎) 을 얻었다. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (59.2 mg).

Figure pat00237
Figure pat00237

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[6-(하이드록시메틸)-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[6-(hydroxymethyl)-9H-purin-9-yl]-2, 10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H -5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis (thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (88.5 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Using the compound (diastereomer 2) (88.5 mg: containing impurities) obtained in step 7 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by purification under the following [purification conditions]. The compound was obtained as the triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 10 ％ - 70 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 10% - 70% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (27.7 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (27.7 mg).

Figure pat00238
Figure pat00238

실시예 18 : CDN18 의 합성 Example 18: Synthesis of CDN18

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-아미노-5-(3-아미노프로필)-7H-피롤로[2,3-d]피리미딘-7-일]-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-amino-5-(3-aminopropyl)-7H-pyrrolo[2,3-d]pyrimidine-7- yl]-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 142][Formula 142]

Figure pat00239
Figure pat00239

[합성 스킴][Synthesis Scheme]

[화학식 143][Formula 143]

Figure pat00240
Figure pat00240

(공정 1) (Process 1)

4-아지드-5-요오드-7-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-7H-피롤로[2,3-d]피리미딘 4-Azide-5-iodo-7-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine

이미 알려진 문헌 (Synth. Co㎜un. 2012, 42, 358-374) 의 4-클로로-5-요오드-7-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-7H-피롤로[2,3-d]피리미딘 (17.96 g) 의 N,N-디메틸포름아미드 (90 ㎖) 용액에, 아지화 테트라부틸암모늄 (10.1 g) 을 첨가하고, 80 ℃ 에서 30 분간 교반하였다. 반응액을 실온으로 되돌리고, 반응액에 포화 탄산수소나트륨 수용액과 아세트산에틸을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (17.66 g) 을 얻었다. 4-Chloro-5-iodine-7-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl of previously known literature (Synth. Commun. 2012, 42, 358-374) To a solution of )-7H-pyrrolo[2,3-d]pyrimidine (17.96 g) in N,N-dimethylformamide (90 mL), tetrabutylammonium azide (10.1 g) was added and stirred at 80°C. Stir for 30 minutes. The reaction solution was returned to room temperature, saturated aqueous sodium hydrogen carbonate solution and ethyl acetate were added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (17.66 g).

Figure pat00241
Figure pat00241

(공정 2) (Process 2)

4-아지드-7-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로파-1-인-1-일]-7H-피롤로[2,3-d]피리미딘 4-Azide-7-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-[3-({[2-(trimethylsilyl)ethoxy]carbonyl} amino) prop-1-yn-1-yl] -7H-pyrrolo[2,3-d] pyrimidine

상기 공정 1 에서 얻어진 화합물 (9.64 g) 의 N,N-디메틸포름아미드 (30 ㎖) - 테트라하이드로푸란 (100 ㎖) 혼합 용액에, 2-(트리메틸실릴)에틸프로파-2-인-1-일카르바메이트 (6.58 g), 트리에틸아민 (4.57 ㎖), 테트라키스(트리페닐포스핀)팔라듐(0) (763 ㎎), 및 요오드화구리(I) (251 ㎎) 을 차례로 첨가하고, 실온에서 밤새 교반하였다. 반응액에 물을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (7.20 g) 을 얻었다. To a mixed solution of the compound (9.64 g) obtained in step 1 above in N,N-dimethylformamide (30 ml) -tetrahydrofuran (100 ml), 2-(trimethylsilyl)ethylprop-2-yn-1- Monocarbamate (6.58 g), triethylamine (4.57 ml), tetrakis(triphenylphosphine)palladium(0) (763 mg), and copper(I) iodide (251 mg) were sequentially added, and was stirred overnight. Water was added to the reaction mixture, and extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (7.20 g).

Figure pat00242
Figure pat00242

(공정 3) (Process 3)

7-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 7-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]- 7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 2 에서 얻어진 화합물 (7.20 g) 의 메탄올 (70 ㎖) - 테트라하이드로푸란 (70 ㎖) 혼합 용액에 20 ％ 수산화팔라듐탄소 (2g) 를 첨가하고, 수소 분위기하, 실온에서 2 시간 교반하였다. 촉매를 여과 제거한 후, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (5.45 g) 을 얻었다.To a methanol (70 ml)-tetrahydrofuran (70 ml) mixed solution of the compound (7.20 g) obtained in step 2 above, 20% palladium hydroxide carbon (2 g) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. After filtering off the catalyst, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (5.45 g).

Figure pat00243
Figure pat00243

(공정 4) (Process 4)

N,N-디벤조일-7-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 N,N-dibenzoyl-7-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-[3-({[2-(trimethylsilyl)ethoxy]carb Bornyl}amino)propyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 3 에서 얻어진 화합물 (5.45 g) 의 디클로로메탄 (60 ㎖) 용액에, 0 ℃ 에서 피리딘 (1.69 ㎖) 과 염화벤조일 (2.01 ㎖) 을 차례로 첨가하고, 0 ℃ 에서 5 분간 교반 후, 추가로 트리에틸아민 (2.91 ㎖) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 디클로로메탄을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 추가로 아세트산에틸과 헥산으로 분말화하였다. 얻어진 고체를 여과 채취하고, 표제 화합물 (6.16 g) 을 얻었다. To a dichloromethane (60 ml) solution of the compound (5.45 g) obtained in step 3 above, pyridine (1.69 ml) and benzoyl chloride (2.01 ml) were added in this order at 0°C, stirred at 0°C for 5 minutes, and then further stirred. Triethylamine (2.91 mL) was added and stirred at room temperature for 2 hours. A saturated aqueous solution of sodium hydrogen carbonate and dichloromethane were added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] and further powdered with ethyl acetate and hexane. The obtained solid was collected by filtration to obtain the title compound (6.16 g).

Figure pat00244
Figure pat00244

(공정 5) (Process 5)

N-벤조일-7-β-D-리보퓨라노실-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 N-benzoyl-7-β-D-ribofuranosyl-5-[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]-7H-pyrrolo[2,3-d ]Pyrimidine-4-amine

상기 공정 4 에서 얻어진 화합물 (6.16 g) 의 테트라하이드로푸란 (250 ㎖) 용액에, 0 ℃ 에서 나트륨메톡시드의 메탄올 용액 (1.0 M, 12.0 ㎖) 을 적하하고, 실온에서 1 시간 교반하였다. 0 ℃ 에서 반응액에 아세트산 (1.07 ㎖) 을 첨가한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [아세트산에틸/메탄올] 로 정제하고, 추가로 아세트산에틸과 헥산으로 분말화하였다. 얻어진 고체를 여과 채취하고, 표제 화합물 (3.35 g) 을 얻었다. To a tetrahydrofuran (250 ml) solution of the compound (6.16 g) obtained in step 4 above, a sodium methoxide solution in methanol (1.0 M, 12.0 ml) was added dropwise at 0°C, followed by stirring at room temperature for 1 hour. After adding acetic acid (1.07 ml) to the reaction solution at 0°C, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate/methanol] and further powdered with ethyl acetate and hexane. The obtained solid was collected by filtration to obtain the title compound (3.35 g).

Figure pat00245
Figure pat00245

(공정 6) (Process 6)

N-벤조일-7-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-리보퓨라노실}-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 N-benzoyl-7-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl}-5-[3-({[2-(trimethylsilyl) Ethoxy]carbonyl}amino)propyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 5 에서 얻어진 화합물 (3.35 g) 을 피리딘으로 공비하고, 잔류물의 무수 피리딘 (50 ㎖) 용액에, 0 ℃ 에서 4,4'-디메톡시트리틸클로라이드 (2.38 g) 를 첨가하고, 실온에서 밤새 교반하였다. 반응액에 에탄올 (5 ㎖) 을 첨가하여 반응을 정지시킨 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고 표제 화합물 (4.34 g) 을 얻었다. The compound (3.35 g) obtained in the above step 5 was azeotroped with pyridine, and 4,4'-dimethoxytrityl chloride (2.38 g) was added to a solution of the residue in anhydrous pyridine (50 ml) at 0°C, followed by stirring at room temperature. Stir overnight. After stopping the reaction by adding ethanol (5 ml) to the reaction solution, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (4.34 g).

Figure pat00246
Figure pat00246

(공정 7) (Process 7)

N-벤조일-7-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 (3'-O-TBS 체) N-benzoyl-7-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}- 5-[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (3'-O-TBS )

상기 공정 6 에서 얻어진 화합물 (4.34 g) 을 사용하여, 실시예 8 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.51 g) 과, 표제 화합물의 위치 이성체인, N-벤조일-7-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 (2'-O-TBS 체) (2.01 g) 을 각각 얻었다.Using the compound (4.34 g) obtained in step 6 above, reaction was carried out in the same manner as in step 4 of Example 8 to obtain the title compound (1.51 g) and the positional isomer of the title compound, N-benzoyl-7-{ 5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-5-[3-({ [2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (2'-O-TBS body) (2.01 g) respectively got it

(3'-OTBS 체) (고극성) (3'-OTBS sieve) (high polarity)

Figure pat00247
Figure pat00247

(2'-OTBS 체) (저극성) (2'-OTBS sieve) (low polarity)

Figure pat00248
Figure pat00248

(공정 8) (Process 8)

N-벤조일-7-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-[tert-부틸(디메틸)실릴]-2-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-5-[3-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)프로필]-7H-피롤로[2,3-d]피리미딘-4-아민 N-benzoyl-7-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-5-[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino) propyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 7 에서 얻어진 화합물 (3'-OTBS 체) (1.51 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 인 원자 상의 디아스테레오머인 표제 화합물의 디아스테레오머 1 (0.77 g) 과 디아스테레오머 2 (0.48 g) 를 각각 얻었다. Using the compound (3'-OTBS substance) (1.51 g) obtained in step 7 above, reaction was carried out in the same manner as in step 6 of Example 1 to obtain diastereomer 1 of the title compound, which is a diastereomer on a phosphorus atom. 0.77 g) and diastereomer 2 (0.48 g) were respectively obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00249
Figure pat00249

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00250
Figure pat00250

(공정 9) (Process 9)

2-(트리메틸실릴)에틸 (3-{4-벤즈아미드-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-7H-피롤로[2,3-d]피리미딘-5-일}프로필)카르바메이트 2-(trimethylsilyl)ethyl (3-{4-benzamide-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2 -Cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}propyl)carb barmate

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.08 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 8 에서 얻어진 화합물 (1.25 g : 디아스테레오머 혼합물) 을 사용하여, 실시예 1 공정 8 및 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 50 ％ (0 분 - 35 분)] 로 인 원자 상의 디아스테레오머를 분리하고, 표제 화합물의 디아스테레오머 1 (0.19 g) 과 디아스테레오머 2 (0.043 g) 를 얻었다. The same reaction as in Example 1 Step 7 was carried out on the following scale (raw material: 1.08 g). A reaction was carried out in the same manner as in Example 1 Step 8 and Example 1 Step 9 using the acetonitrile solution of the obtained compound and the compound obtained in Step 8 (1.25 g: diastereomer mixture), and the title compound was obtained by phosphorus Obtained as a diastereomer mixture on atoms. The diastereomer on the phosphorus atom was separated by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 50% (0 min - 35 min)], diastereomer 1 of the title compound (0.19 g) and diastereomer 2 (0.043 g) were obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1419(M＋H)＋. MS(ESI)m/z: 1419(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1419(M＋H)＋.MS(ESI)m/z: 1419(M+H) + .

(공정 10-1) (Process 10-1)

2-(트리메틸실릴)에틸 (3-{4-아미노-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-7H-피롤로[2,3-d]피리미딘-5-일}프로필)카르바메이트 2-(trimethylsilyl)ethyl (3-{4-amino-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl [cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecin-7-yl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}propyl)carbamate

상기 공정 9 에서 얻어진 화합물 (디아스테레오머 1) (0.19 g) 을 사용하여, 실시예 12 공정 8-1 과 동일한 수법으로 반응을 실시하였다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 40 ％ - 70 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물 (58 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 12 Step 8-1 using the compound (diastereomer 1) (0.19 g) obtained in Step 9 above. Purification was performed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 40% - 70% (0 min - 35 min)] to obtain the title compound (58 mg).

MS(ESI)m/z : 1158(M＋H)＋.MS(ESI)m/z: 1158(M+H) + .

(공정 10-2) (Process 10-2)

2-(트리메틸실릴)에틸 (3-{4-아미노-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-7H-피롤로[2,3-d]피리미딘-5-일}프로필)카르바메이트 2-(trimethylsilyl)ethyl (3-{4-amino-7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl [cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecin-7-yl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}propyl)carbamate

상기 공정 9 에서 얻어진 화합물 (디아스테레오머 2) (43 ㎎) 을 사용하여, 실시예 12 공정 8-1 과 동일한 수법으로 반응을 실시하였다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 60 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물 (15 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 12 Step 8-1 using the compound (diastereomer 2) (43 mg) obtained in Step 9 above. Purification was performed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 60% (0 min - 35 min)] to obtain the title compound (15 mg).

MS(ESI)m/z : 1158(M＋H)＋.MS(ESI)m/z: 1158(M+H) + .

(공정 11-1) (Process 11-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-아미노-5-(3-아미노프로필)-7H-피롤로[2,3-d]피리미딘-7-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-amino-5-(3-aminopropyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3, 5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 10-1 에서 얻어진 화합물 (58 ㎎) 의 테트라하이드로푸란 (1㎖) 용액에, 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 5 ㎖) 을 첨가하고, 실온에서 밤새 교반하였다. 반응이 완결되어 있지 않기 때문에, 추가로 40 ℃ 에서 3 시간 교반하였다. 반응액을 감압 농축 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 40 ％ (0 분 - 35 분)] 로 정제하였다. 또한 Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 30 ％] 로 정제하고, 표제 화합물 (25 ㎎) 을 얻었다. To a tetrahydrofuran (1 ml) solution of the compound (58 mg) obtained in step 10-1 above, a tetrahydrofuran solution of tetrabutylammonium fluoride (about 1 M, 5 ml) was added, and the mixture was stirred at room temperature overnight. Since the reaction was not completed, it was further stirred at 40°C for 3 hours. The reaction solution was concentrated under reduced pressure and purified by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 40% (0 min - 35 min)]. Further purification was performed with Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/acetonitrile, acetonitrile: 0% - 30%] to obtain the title compound (25 mg).

MS(ESI)m/z : 786(M＋H)＋.MS(ESI) m/z : 786(M+H) + .

(공정 11-2) (Process 11-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-아미노-5-(3-아미노프로필)-7H-피롤로[2,3-d]피리미딘-7-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-amino-5-(3-aminopropyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3, 5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 10-2 에서 얻어진 화합물 (15 ㎎) 을 사용하여, 상기 공정 11-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (7.2 ㎎) 을 얻었다. Using the compound (15 mg) obtained in step 10-2 above, a reaction was carried out in the same manner as in step 11-1 to obtain the title compound (7.2 mg).

MS(ESI)m/z : 786(M＋H)＋.MS(ESI) m/z : 786(M+H) + .

(공정 12-1) (Process 12-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-아미노-5-(3-아미노프로필)-7H-피롤로[2,3-d]피리미딘-7-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-amino-5-(3-aminopropyl)-7H-pyrrolo[2,3-d]pyrimidine- 7-yl] -15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 11-1 에서 얻어진 화합물 (25 ㎎) 을 사용하여, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (17 ㎎) 을 얻었다. Using the compound (25 mg) obtained in step 11-1 above, salt exchange was carried out in the same manner as [Conversion to sodium salt] described in step 11 of Example 1 to obtain the title compound (17 mg).

Figure pat00251
Figure pat00251

(공정 12-2) (Process 12-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-아미노-5-(3-아미노프로필)-7H-피롤로[2,3-d]피리미딘-7-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[4-amino-5-(3-aminopropyl)-7H-pyrrolo[2,3-d]pyrimidine- 7-yl] -15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 11-2 에서 얻어진 화합물 (7.2 ㎎) 을 사용하여, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (6.0 ㎎) 을 얻었다. Using the compound (7.2 mg) obtained in step 11-2 above, salt exchange was carried out in the same manner as [Conversion to sodium salt] described in step 11 of Example 1 to obtain the title compound (6.0 mg).

Figure pat00252
Figure pat00252

실시예 19 : CDN19 의 합성 Example 19: Synthesis of CDN19

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) )-14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H- 5,8-methano- 2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 144][Formula 144]

Figure pat00253
Figure pat00253

[합성 스킴][Synthesis Scheme]

[화학식 145][Formula 145]

Figure pat00254
Figure pat00254

(공정 1) (Process 1)

3-브로모-N-(프로파-2-엔-1-일)-1H-피라졸로[3,4-d]피리미딘-4-아민 3-Bromo-N-(prop-2-en-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

시판 (Bepharm) 되는 3-브로모-4-클로로-1H-피라졸로[3,4-d]피리미딘 (8.50 g) 을 디옥산 (140 ㎖) 에 현탁시키고, 실온에서 N,N,-디이소프로필에틸아민 (12.5 ㎖) 과 알릴아민 (11 ㎖) 을 첨가하고, 동 온도에서 70 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 디클로로메탄과 아세트산에틸로 슬러리상으로 한 후, 고체를 여과 채취하였다 (고체 1). 여과액을 감압 농축 후, 동일한 조작을 2 회 반복해서 각각 고체 2 와 고체 3 을 얻었다. 마지막 여과액을 실리카 겔 칼럼 [헥산/아세트산에틸] 로 정제하여 고체 4 를 얻었다. 또한, 고체 1 을 실리카 겔 칼럼 [헥산/아세트산에틸] 로 정제하여 고체 5 를 얻었다. 고체 4 와 고체 5 를 아울러, 표제 화합물 (4.48 g) 을 얻었다. 고체 2 와 고체 3 을 아울러, 불순물을 포함하는 표제 화합물 (3.78 g) 을 얻었다. Commercially available (Bepharm) 3-bromo-4-chloro-1H-pyrazolo[3,4-d]pyrimidine (8.50 g) was suspended in dioxane (140 ml), and N,N,-di Isopropylethylamine (12.5 ml) and allylamine (11 ml) were added, and the mixture was stirred at the same temperature for 70 hours. The reaction solution was concentrated under reduced pressure, and the residue was obtained as a slurry with dichloromethane and ethyl acetate, and the solid was collected by filtration (solid 1). After concentration of the filtrate under reduced pressure, the same operation was repeated twice to obtain a solid 2 and a solid 3, respectively. The final filtrate was purified with a silica gel column [hexane/ethyl acetate] to obtain a solid 4. Further, solid 1 was purified with a silica gel column [hexane/ethyl acetate] to obtain solid 5. Solid 4 and Solid 5 were combined to obtain the title compound (4.48 g). Solid 2 and Solid 3 were combined to obtain the title compound (3.78 g) containing impurities.

Figure pat00255
Figure pat00255

(공정 2) (Process 2)

3-브로모-N-(프로파-2-엔-1-일)-1-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-1H-피라졸로[3,4-d]피리미딘-4-아민 3-Bromo-N-(prop-2-en-1-yl)-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[ 3,4-d] pyrimidin-4-amine

상기 공정 1 에서 얻어진 화합물 (1.00 g) 과 시판 (Ark Pharm) 되는 1-O-아세틸-2,3,5-트리-O-벤조일-β-D-리보퓨라노스 (2.58 g) 를 니트로메탄 (50 ㎖) 에 현탁시킨 후, 가열하여 용해시켰다. 가열 환류하에서 3불화붕소디에틸에테르 착물 (0.63 ㎖) 을 첨가하고, 동 온도에서 1 시간 교반하였다. 1-O-아세틸-2,3,5-트리-O-벤조일-β-D-리보퓨라노스 (0.40 g) 와 3불화붕소디에틸에테르 착물 (0.10 ㎖) 을 추가하여, 3 시간 더 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.80 g) 을 얻었다. The compound obtained in step 1 (1.00 g) and commercially available (Ark Pharm) 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (2.58 g) were mixed with nitromethane ( 50 ml), and then heated to dissolve. Boron trifluoride diethyl ether complex (0.63 ml) was added under heating under reflux, and the mixture was stirred at the same temperature for 1 hour. 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (0.40 g) and boron trifluoride diethyl ether complex (0.10 ml) were added, and the mixture was stirred for another 3 hours. . After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.80 g).

Figure pat00256
Figure pat00256

(공정 3) (Process 3)

3-에테닐-N-(프로파-2-엔-1-일)-1-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-1H-피라졸로[3,4-d]피리미딘-4-아민 3-ethenyl-N-(prop-2-en-1-yl)-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[ 3,4-d] pyrimidin-4-amine

상기 공정 2 에서 얻어진 화합물 (11.65 g) 의 톨루엔 (120 ㎖) 에 용액을, 감압하에서 초음파 처리하여 탈기하였다. 질소 분위기하, 반응액에 트리부틸비닐주석 (12.8 ㎖) 과 테트라키스(트리페닐포스핀)팔라듐(0) (2.73 g) 을 첨가하고, 2 시간 가열 환류하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/아세트산에틸] 로 정제하고, 표제 화합물 (10.07 g) 을 얻었다. A solution of the compound (11.65 g) obtained in the above step 2 in toluene (120 ml) was treated with ultrasonic waves under reduced pressure and degassed. Tributylvinyltin (12.8 ml) and tetrakis(triphenylphosphine)palladium(0) (2.73 g) were added to the reaction solution under a nitrogen atmosphere, and the mixture was heated to reflux for 2 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [dichloromethane/ethyl acetate] to obtain the title compound (10.07 g).

Figure pat00257
Figure pat00257

(공정 4) (Process 4)

2-(2,3,5-트리-O-벤조일-β-D-리보퓨라노실)-6,7-디하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 2-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-6,7-dihydro-2H-1,2,3,5,6-pentaazabenzo [cd] Azulene

상기 공정 3 에서 얻어진 화합물 (9.0 g) 을 벤젠으로 2 회 공비하였다. 잔류물의 디클로로메탄 용액 (480 ㎖) 에 (＋)-10-캠퍼술폰산 (4.2 g) 과 벤질리덴[1,3-비스(2,4,6-트리메틸페닐)이미다졸리딘-2-일리덴]디클로라이드(트리시클로헥실-λ5-포스파닐)루테늄 (제 2 세대 Grubbs 촉매) (360 ㎎) 을 첨가하고, 3 시간 가열 환류하였다. 제 2 세대 Grubbs 촉매 (360 ㎎) 를 추가하고, 1 시간 더 가열 환류하였다. 반응액을 실온까지 냉각시키고, 포화 탄산수소나트륨 수용액과 포화 식염수로 차례로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (6.39 g) 을 얻었다.The compound (9.0 g) obtained in step 3 was azeotroped twice with benzene. (+)-10-camphorsulfonic acid (4.2 g) and benzylidene[1,3-bis(2,4,6-trimethylphenyl)imidazolidin-2-ylidene [1,3-bis(2,4,6-trimethylphenyl)imidazolidin-2-ylidene ]Dichloride(tricyclohexyl-λ 5 -phosphanyl)ruthenium (second generation Grubbs catalyst) (360 mg) was added, and the mixture was heated to reflux for 3 hours. Second generation Grubbs catalyst (360 mg) was added and heated to reflux for another hour. The reaction solution was cooled to room temperature, washed sequentially with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (6.39 g).

Figure pat00258
Figure pat00258

(공정 5) (Process 5)

2-β-D-리보퓨라노실-6,7-디하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 2-β-D-ribofuranosyl-6,7-dihydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (620 ㎎) 의 메탄올 (10 ㎖) - 테트라하이드로푸란 (5.0 ㎖) 혼합 용액에, 실온에서 나트륨메톡시드의 메탄올 용액 (1.0 M, 0.10 ㎖) 을 첨가하고, 동 온도에서 18 시간 교반하였다. 반응액을 1 규정 염산으로 중화 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (279 ㎎) 을 얻었다. To a methanol (10 ml)-tetrahydrofuran (5.0 ml) mixed solution of the compound (620 mg) obtained in step 4 above, a methanol solution of sodium methoxide (1.0 M, 0.10 ml) was added at room temperature, and Stirred for 18 hours. The reaction solution was neutralized with 1 normal hydrochloric acid and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (279 mg).

Figure pat00259
Figure pat00259

(공정 6) (Process 6)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-6,7-디하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7-dihydro -2H-1,2,3,5,6-pentaazabenzo[cd]azulene

상기 공정 5 에서 얻어진 화합물 (2.81 g) 을 사용하여, 실시예 1 의 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.72 g) 을 얻었다. Using the compound (2.81 g) obtained in the above step 5, a reaction was carried out in the same manner as in step 1 of Example 1 to obtain the title compound (4.72 g).

Figure pat00260
Figure pat00260

(공정 7) (Process 7)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8, 9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulene

상기 공정 6 에서 얻어진 화합물 (2.12 g) 의 테트라하이드로푸란 (20 ㎖) 용액에, 아세트산 (파스퇴르 피펫으로 3 방울), 10 ％ 팔라듐탄소 (AD) wet (0.82 g) 를 첨가하고, 수소 분위기하, 실온에서 3 시간 교반하였다. 촉매를 여과 제거한 후, 테트라하이드로푸란으로 세정하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.09 g) 을 얻었다. Acetic acid (3 drops with a Pasteur pipette) and 10% palladium carbon (AD) wet (0.82 g) were added to a tetrahydrofuran (20 ml) solution of the compound (2.12 g) obtained in step 6 above, and then under a hydrogen atmosphere, It was stirred at room temperature for 3 hours. After filtering off the catalyst, it was washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (2.09 g).

Figure pat00261
Figure pat00261

(공정 8) (Process 8)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6, 7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulene

상기 공정 7 에서 얻어진 화합물 (2.09 g) 을 사용하여, 실시예 1 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.23 g) 을 얻었다. Using the compound (2.09 g) obtained in the above step 7, a reaction was carried out in the same manner as in Example 1 step 4 to obtain the title compound (2.23 g).

Figure pat00262
Figure pat00262

(공정 9) (Process 9)

6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}- 6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulene

상기 공정 8 에서 얻어진 화합물 (2.23 g) 을 사용하여, 실시예 1 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.69 g) 을 얻었다. Using the compound (2.23 g) obtained in the above step 8, a reaction was carried out in the same manner as in Example 1 step 5 to obtain the title compound (2.69 g).

Figure pat00263
Figure pat00263

(공정 10) (Process 10)

6-벤조일-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌 6-benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-1,2,3,5,6 -pentaazabenzo[cd]azulene

상기 공정 9 에서 얻어진 화합물 (2.69 g) 을 사용하여, 실시예 5 의 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.93 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. Using the compound (2.69 g) obtained in step 9 above, a reaction was conducted in the same manner as in step 4 of Example 5 to obtain the title compound (2.93 g) on a phosphorus atom-phase diastereomer mixture (diastereomer ratio = 6: 4) was obtained as

Figure pat00264
Figure pat00264

(공정 11) (Process 11)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy } -2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

상기 공정 10 에서 얻어진 화합물 (1.04 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3-O-(디하이드록시포스파닐)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 시판 (Cool Pharm) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.20 g) 을 사용하여, 실시예 1 공정 8, 실시예 1 공정 9, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 - 50 ％ (0 분 - 35 분)] 로 인 원자 상의 디아스테레오머를 분리하고, 표제 화합물의 디아스테레오머 1 (15 ㎎) 과 디아스테레오머 2 (55 ㎎) 를 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). Using the compound (1.04 g) obtained in step 10 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3 -O-(dihydroxyphosphanyl)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo [cd] A solution of azulene in acetonitrile was obtained. This acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]- Using 2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.20 g), Example 1 Step 8, Example 1 Step 9, Example 1 Step 9, The reaction was carried out in the same manner as in Example 1 Step 10 to obtain the title compound as a mixture of diastereomers on a phosphorus atom. The diastereomer on the phosphorus atom was separated by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 25 - 50% (0 min - 35 min)] to obtain diastereomer 1 of the title compound (15 mg ) and diastereomer 2 (55 mg) were obtained (retention time by HPLC: diastereomer 1 > 2).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 959(M＋H)＋. MS(ESI) m/z : 959(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 959(M＋H)＋.MS(ESI) m/z : 959(M+H) + .

(공정 12-1) (Process 12-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16- Dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 1) (20 ㎎) 에, 트리에틸아민3불화수소산염 (1㎖) 을 첨가하고, 45 ℃ 에서 2 시간 교반하였다. 반응액을 빙랭한 1 M 탄산수소트리에틸암모늄 수용액 (3 ㎖) 과 트리에틸아민 (1㎖) 의 혼합 용액에 첨가하여 반응을 정지시켰다. Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민수/아세토니트릴, 아세토니트릴 : 0 ％ - 17 ％] 로 정제하고, 표제 화합물 (15 ㎎) 을 얻었다. To the compound (diastereomer 1) (20 mg) obtained in step 11 above, triethylamine hydrofluoric acid salt (1 ml) was added, and the mixture was stirred at 45°C for 2 hours. The reaction solution was added to an ice-cooled 1 M aqueous solution of triethylammonium hydrogen carbonate (3 ml) and a mixed solution of triethylamine (1 ml) to stop the reaction. Purification was performed with Sep-Pak (registered trademark) C18 [0.1% aqueous triethylamine/acetonitrile, acetonitrile: 0% - 17%] to obtain the title compound (15 mg).

MS(ESI)m/z : 731(M＋H)＋.MS(ESI) m/z : 731(M+H) + .

(공정 12-2) (Process 12-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16- Dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 2) (43 ㎎) 을 사용하여, 상기 공정 12-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (34 ㎎) 을 얻었다. Using the compound (diastereomer 2) (43 mg) obtained in step 11 above, a reaction was carried out in the same manner as in step 12-1 to obtain the title compound (34 mg).

MS(ESI)m/z : 731(M＋H)＋.MS(ESI) m/z : 731(M+H) + .

(공정 13-1) (Process 13-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5 ,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiol rate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 12-1 에서 얻어진 화합물 (15 ㎎) 을 사용하여, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물의 디아스테레오머 혼합물 (12 ㎎) 을 얻었다. Using the compound (15 mg) obtained in step 12-1 above, salt exchange was carried out in the same manner as [Conversion to sodium salt] described in step 11 of Example 1, and a diastereomer mixture of the title compound (12 mg ) was obtained.

Figure pat00265
Figure pat00265

(공정 13-2) (Process 13-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-1,2,3,5,6-펜타아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-1,2,3,5,6-pentaazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5 ,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiol rate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 12-2 에서 얻어진 화합물 (34 ㎎) 을 사용하여, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물의 디아스테레오머 혼합물 (28 ㎎ : 디아스테레오머비 = 4 : 1) 을 얻었다.Using the compound (34 mg) obtained in step 12-2 above, salt exchange was carried out in the same manner as [Conversion to sodium salt] described in step 11 of Example 1, and a diastereomer mixture of the title compound (28 mg : Diastereomer ratio = 4: 1) was obtained.

Figure pat00266
Figure pat00266

실시예 20 : CDN20 의 합성 Example 20: Synthesis of CDN20

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-oxa-2; 3,5-triazabenzo[cd]azulen-2(7H)-yl)-15,16-dihydroxy-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 146][Formula 146]

Figure pat00267
Figure pat00267

[합성 스킴][Synthesis Scheme]

[화학식 147][Formula 147]

Figure pat00268
Figure pat00268

(공정 1) (Process 1)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-4-클로로-5-요오드-7H-피롤로[2,3-d]피리미딘 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-4-chloro-5- Iodine-7H-pyrrolo[2,3-d]pyrimidine

이미 알려진 문헌 (J. Med. Chem. 2008, 51, 3934-3945) 의 4-클로로-5-요오드-7-β-D-리보퓨라노실-7H-피롤로[2,3-d]피리미딘 (3.15 g) 을 사용하여, 실시예 1 의 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.97 g) 을 얻었다. 4-chloro-5-iodine-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrrole of the previously known literature (J. Med. Chem. 2008, 51, 3934-3945) The reaction was carried out in the same manner as in step 1 of Example 1 using midine (3.15 g) to obtain the title compound (2.97 g).

Figure pat00269
Figure pat00269

(공정 2) (Process 2)

4-(벤질옥시)-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-요오드-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl} -5-iodo-7H-pyrrolo[2,3-d]pyrimidine

상기 공정 1 에서 얻어진 화합물 (4.97 g) 과 벤질알코올 (1.0 ㎖) 의 테트라하이드로푸란 (50 ㎖) 용액에, 빙랭하에서 수소화나트륨 (미네랄 오일을 37 ％ 함유) (426 ㎎) 을 첨가하고, 실온으로 승온하여 밤새 교반하였다. 빙랭하, 반응액에 포화 염화암모늄 수용액을 첨가하여 반응을 정지시켰다. 반응액을 아세트산에틸로 추출 후, 유기층을 물, 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (3.38 g) 을 얻었다. To a tetrahydrofuran (50 ml) solution of the compound (4.97 g) obtained in step 1 and benzyl alcohol (1.0 ml), sodium hydride (containing 37% mineral oil) (426 mg) was added under ice-cooling, and cooled to room temperature. The temperature was raised and stirred overnight. Under ice-cooling, a saturated ammonium chloride aqueous solution was added to the reaction mixture to stop the reaction. After extracting the reaction solution with ethyl acetate, the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (3.38 g).

Figure pat00270
Figure pat00270

(공정 3) (Process 3)

4-(벤질옥시)-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(3-하이드록시프로파-1-인-1-일)-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl} -5-(3-hydroxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine

상기 공정 2 에서 얻어진 화합물 (3.38 g) 과 2-프로핀-1-올 (1.35 ㎖) 을 사용하고, 반응 온도를 실온으로 하여, 실시예 1 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.22 g) 을 얻었다. The reaction was carried out in the same manner as in Example 1 Step 2 using the compound (3.38 g) obtained in Step 2 above and 2-propyn-1-ol (1.35 mL) at room temperature, and the reaction was carried out to obtain the title compound. (2.22 g) was obtained.

Figure pat00271
Figure pat00271

(공정 4) (Process 4)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(3-하이드록시프로필)-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-온 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(3-hydride Roxypropyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

상기 공정 3 에서 얻어진 화합물 (3.38 g) 을 사용하여, 반응 용매를 메탄올 (20 ㎖) - 테트라하이드로푸란 (20 ㎖) 의 혼합액으로 하고, 실시예 19 공정 7 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (0.92 g) 을 얻었다. Using the compound (3.38 g) obtained in the above step 3, the reaction solvent was a mixture of methanol (20 ml) and tetrahydrofuran (20 ml), and the reaction was carried out in the same manner as in Example 19 step 7, A compound (0.92 g) was obtained.

Figure pat00272
Figure pat00272

(공정 5) (Process 5)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-2,7,8, 9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (0.92 g) 의 테트라하이드로푸란 (32 ㎖) 용액에, 빙랭하에서 트리페닐포스핀 (0.62 g) 과 아조디카르복실산디이소프로필 (0.47 ㎖) 을 첨가하고, 실온까지 승온하여 1 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (0.77 g) 을 얻었다. To a solution of the compound (0.92 g) obtained in step 4 above in tetrahydrofuran (32 ml), triphenylphosphine (0.62 g) and diisopropyl azodicarboxylic acid (0.47 ml) were added under ice-cooling, and the temperature was raised to room temperature. and stirred for 1 hour. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (0.77 g).

Figure pat00273
Figure pat00273

(공정 6) (Process 6)

2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-2,7, 8,9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulene

상기 공정 5 에서 얻어진 화합물 (0.95 g) 을 사용하여, 실시예 1 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.13 g) 을 얻었다. Using the compound (0.95 g) obtained in step 5 above, reaction was carried out in the same manner as in step 5 of Example 1 to obtain the title compound (1.13 g).

Figure pat00274
Figure pat00274

(공정 7) (Process 7)

2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd ]azulene

상기 공정 6 에서 얻어진 화합물 (1.13 g) 을 사용하여, 정제에 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 을 사용한 것 이외는, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.00 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다.Using the compound (1.13 g) obtained in the above step 6, the reaction was carried out in the same manner as in Example 5 step 4, except that silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] was used for purification. carried out to obtain the title compound (1.00 g) as a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 6:4).

Figure pat00275
Figure pat00275

(공정 8) (Process 8)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoe Toxy)-14-(8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-2-oxo-2-sulfanyl-10 -sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 7 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-O-[tert-부틸(디메틸)실릴]-3-O-(디하이드록시포스파닐)-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 시판 (Cool Pharm) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.28 g) 을 사용하여, 실시예 1 공정 8, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 을 포함하는 혼합물과 디아스테레오머 2 를 포함하는 혼합물을 각각 얻었다. Using the compound (1.00 g) obtained in step 7 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-( Dihydroxyphosphanyl)-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulene in acetonitrile solution got This acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]- Using 2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.28 g), the same steps as Example 1 Step 8 and Example 1 Step 9 The reaction was carried out by the method to obtain a mixture containing diastereomer 1 and a mixture containing diastereomer 2 of the title compound, respectively.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1116(M＋H)＋. MS(ESI)m/z: 1116(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1116(M＋H)＋.MS(ESI)m/z: 1116(M+H) + .

(공정 9-1) (Process 9-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16- Bis{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl) -2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 1 을 포함하는 혼합물) 을 전량 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (73 ㎎) 을 트리에틸아민염으로서 얻었다. After reacting in the same manner as in Example 1 Step 10 using the entire amount of the compound (mixture containing diastereomer 1) obtained in Step 8 above, purification was carried out under the following [Purification conditions], and the title compound ( 73 mg) as triethylamine salt.

[정제 조건] HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 60 ％ (0 분 - 35 분)]. [Purification conditions] HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 60% (0 min - 35 min)].

MS(ESI)m/z : 959(M＋H)＋.MS(ESI) m/z : 959(M+H) + .

(공정 9-2) (Process 9-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16- Bis{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl) -2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 2 를 포함하는 혼합물) 을 전량 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (58 ㎎) 을 트리에틸아민염으로서 얻었다. After carrying out the reaction in the same manner as in Example 1 Step 10 using the entire amount of the compound (mixture containing diastereomer 2) obtained in Step 8 above, purification was carried out under the following [Purification conditions], and the title compound ( 58 mg) as triethylamine salt.

[정제 조건] HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 50 ％ (0 분 - 35 분)]. [Purification conditions] HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 25% - 50% (0 min - 35 min)].

MS(ESI)m/z : 959(M＋H)＋.MS(ESI) m/z : 959(M+H) + .

(공정 10-1) (Process 10-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-oxa- 2,3,5-triazabenzo[cd]azulene-2(7H)-yl)-15,16-dihydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8- Methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 9-1 에서 얻어진 화합물 (68 ㎎) 을 사용하여, 실시예 19 공정 12-1 과 동일한 수법으로 반응을 실시한 후, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (42 ㎎) 을 얻었다. Using the compound (68 mg) obtained in step 9-1 above, reaction was carried out by the same method as in Example 19 step 12-1, followed by the same method as [Conversion to sodium salt] described in Example 1 step 11 Salt exchange was carried out to obtain the title compound (42 mg).

Figure pat00276
Figure pat00276

(공정 10-2) (Process 10-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-oxa- 2,3,5-triazabenzo[cd]azulene-2(7H)-yl)-15,16-dihydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8- Methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 9-2 에서 얻어진 화합물 (58 ㎎) 을 사용하여, 실시예 19 공정 12-1 과 동일한 수법으로 반응을 실시한 후, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (35 ㎎) 을 얻었다. Using the compound (58 mg) obtained in the above step 9-2, the reaction was carried out by the same method as in Example 19 step 12-1, followed by the same method as [conversion to sodium salt] described in Example 1 step 11 Salt exchange was carried out to obtain the title compound (35 mg).

Figure pat00277
Figure pat00277

실시예 21 : 약물 링커 1 의 합성 Example 21: Synthesis of Drug Linker 1

[합성 스킴][Synthesis Scheme]

[화학식 148][Formula 148]

Figure pat00278
Figure pat00278

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-페닐알라닐글리신트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-phenylalanylglycinetrifluoroacetate

시판 (BACHEM) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-페닐알라닐글리신 (3.00 g) 의 디클로로메탄 (30 ㎖) 용액에 실온에서 트리플루오로아세트산 (15 ㎖) 을 첨가하고, 동 온도에서 3 시간 교반하였다. 반응액을 감압 농축 후, 톨루엔에 현탁시켜 재차 감압 농축하였다. 이 농축 조작을 추가로 2 회 반복하였다. 잔류물을 디에틸에테르 (100 ㎖) 로 슬러리상으로 한 후, 여과 채취하고, 표제 화합물 (3.27 g) 의 미정제체를 얻었다. Trifluoroacetic acid (15 ml) was added to a dichloromethane (30 ml) solution of commercially available (BACHEM) N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (3.00 g) at room temperature. It was added and stirred at the same temperature for 3 hours. After the reaction solution was concentrated under reduced pressure, it was suspended in toluene and again concentrated under reduced pressure. This concentration operation was repeated a further two times. The residue was made into a slurry with diethyl ether (100 mL) and collected by filtration to obtain a crude product of the title compound (3.27 g).

Figure pat00279
Figure pat00279

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐글리신 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanylglycine

상기 공정 1 에서 얻어진 화합물 (2.09 g) 의 N,N-디메틸포르미아미드 (46.4 ㎖) 용액에 트리에틸아민 (0.804 ㎖) 과 1-{[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]옥시}피롤리딘-2,5-디온 (1.87 g) 을 첨가하고, 실온에서 21 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하였다. 얻어진 화합물의 디클로로메탄 용액에 디에틸에테르를 첨가하여 슬러리상으로 한 후, 여과 채취하고, 표제 화합물 (2.10 g) 을 얻었다. Triethylamine (0.804 ml) and 1-{[4-(11,12-didehydrodibenzo[b ,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (1.87 g) was added, and the mixture was stirred at room temperature for 21 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane/methanol]. After adding diethyl ether to a dichloromethane solution of the obtained compound to form a slurry, the mixture was collected by filtration to obtain the title compound (2.10 g).

Figure pat00280
Figure pat00280

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐글리시네이트 2,5-dioxopyrrolidin-1-ylN-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanylglycinate

상기 공정 2 에서 얻어진 화합물 (2.10 g) 의 N,N-디메틸포르미아미드 (33.7 ㎖) 용액에 N-하이드록시숙신이미드 (426 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (710 ㎎) 을 첨가하고, 질소 분위기하, 실온에서 16 시간 교반하였다. 반응액을 디클로로메탄으로 희석 후, 빙수로 3 회 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 유상 (油狀) 의 잔류물에 아세트산에틸을 첨가하여 고체를 석출시켰다. 용매를 감압하에서 증류 제거, 얻어진 고체에 디에틸에테르를 첨가하여 슬러리상으로 한 후, 여과 채취하고, 표제 화합물 (2.18 g) 을 얻었다.To a solution of the compound (2.10 g) obtained in step 2 above in N,N-dimethylformiamide (33.7 ml), N-hydroxysuccinimide (426 mg) and 1-ethyl-3-(3-dimethylaminopropyl) - Carbodiimide hydrochloride (710 mg) was added, and the mixture was stirred for 16 hours at room temperature under a nitrogen atmosphere. After diluting the reaction solution with dichloromethane, it was washed three times with ice water and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the oily residue to deposit a solid. The solvent was distilled off under reduced pressure, and diethyl ether was added to the obtained solid to form a slurry, which was collected by filtration to obtain the title compound (2.18 g).

Figure pat00281
Figure pat00281

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo -2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H; 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7- yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)glycinamide

(약물 링커 1) (drug linker 1)

실시예 5 공정 8-2 에서 얻어진 화합물 (10.0 ㎎) 의 N,N-디메틸포름아미드 (1㎖) 용액에 트리에틸아민 (8 ㎕) 과 상기 공정 3 에서 얻어진 화합물 (17.6 ㎎) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에, 벤질아민 (3 ㎕) 을 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 10 mM 아세트산트리에틸암모늄 수용액과 메탄올을 첨가하고, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 50 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (10.9 ㎎) 을 얻었다.Example 5 To a solution of the compound (10.0 mg) obtained in step 8-2 in N,N-dimethylformamide (1 ml) was added triethylamine (8 µl) and the compound obtained in step 3 (17.6 mg), It was stirred for 2 hours at room temperature. To the reaction solution, benzylamine (3 µl) was added, and the mixture was stirred at room temperature for 1 hour. 10 mM triethylammonium acetate aqueous solution and methanol were added to the reaction solution, and C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, aceto nitrile: 5% - 50% (0 min - 40 min)] to obtain the title compound (10.9 mg).

Figure pat00282
Figure pat00282

실시예 22 : 약물 링커 2 의 합성 Example 22: Synthesis of Drug Linker 2

[합성 스킴][Synthesis Scheme]

[화학식 149][Formula 149]

Figure pat00283
Figure pat00283

(공정 1) (Process 1)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine

이노신 (10.0 g) 의 피리딘 (50 ㎖) 과 N,N-디메틸아세트아미드 (50 ㎖) 용액에, 4,4'-디메톡시트리틸클로라이드 (15.2 g) 를 0 ℃ 에서 첨가한 후, 4 ℃ 에서 64 시간 교반하였다. 반응액에 메탄올 (2 ㎖) 을 첨가하고, 10 분 교반 후에 50 ㎖ 정도까지 농축하였다. 잔류물에 2-브로모에틸벤조에이트 (7.02 ㎖) 와 2,3,4,6,7,8,9,10-옥타하이드로피리미드[1,2-a]아제핀 (13.9 ㎖) 을 첨가하고, 실온에서 1 일 교반하였다. 반응액에, 포화 탄산수소나트륨 수용액과 물을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (15.2 g) 을 얻었다. To a solution of inosine (10.0 g) in pyridine (50 mL) and N,N-dimethylacetamide (50 mL), 4,4'-dimethoxytrityl chloride (15.2 g) was added at 0 °C, followed by 4 °C. was stirred for 64 hours. Methanol (2 ml) was added to the reaction solution, and after stirring for 10 minutes, the mixture was concentrated to about 50 ml. 2-Bromoethylbenzoate (7.02 mL) and 2,3,4,6,7,8,9,10-octahydropyrimide[1,2-a]azepine (13.9 mL) were added to the residue. and stirred at room temperature for 1 day. To the reaction solution, a saturated aqueous solution of sodium hydrogen carbonate and water were added, followed by extraction with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (15.2 g).

Figure pat00284
Figure pat00284

(공정 2) (Process 2)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]inosine

상기 공정 1 에서 얻어진 화합물 (3.01 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 합성을 실시하고, 표제 화합물 (1.20 g) 과 표제 화합물의 위치 이성체인 1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]이노신 (1.22 g) 을 얻었다. Using the compound (3.01 g) obtained in the above step 1, synthesis was carried out in the same manner as in Example 5 step 3 to obtain the title compound (1.20 g) and 1-[2-(benzoyloxy), which is a positional isomer of the title compound. This gave ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]inosine (1.22 g).

Figure pat00285
Figure pat00285

(2'-O-TBS 체) (2'-O-TBS sieve)

Figure pat00286
Figure pat00286

(공정 3) (Process 3)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'- O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine

상기 공정 2 에서 얻어진 화합물 (1.20 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 합성을 실시하고, 표제 화합물 (1.41 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 0.55 : 0.45) 로서 얻었다.Using the compound (1.20 g) obtained in step 2 above, synthesis was carried out in the same manner as in step 4 of Example 5, and the title compound (1.41 g) was obtained by preparing a diastereomer mixture on a phosphorus atom (diastereomer ratio = 0.55 : 0.45). ) was obtained as

Figure pat00287
Figure pat00287

(공정 4) (Process 4)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.40 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 3 에서 얻어진 화합물 (1.41 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 1.40 g). The reaction was carried out in the same manner as in Example 1 Step 8 using the acetonitrile solution of the obtained compound and the compound (1.41 g) obtained in the step 3 above. The obtained crude product was used as it was for the next reaction.

(공정 5) (Process 5)

2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 4 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (778 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 4 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (778 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1264(M＋H)＋.MS(ESI) m/z : 1264(M+H) + .

(공정 6) (Process 6)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- [1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetra hydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 5 에서 얻어진 화합물 (778 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (255 ㎎) 과 디아스테레오머 2 (불순물 함유) 를 얻었다. 디아스테레오머 2 를 분취 HPLC [물/0.2 ％ 트리에틸아민 함유 아세토니트릴, 0.2 ％ 트리에틸아민 함유 아세토니트릴 : 5 ％ - 50 ％ (0 분 - 40 분)] 로 재차 정제하고, 표제 화합물의 디아스테레오머 2 (94.6 ㎎) 를 얻었다. Using the compound (778 mg) obtained in step 5 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (255 mg) and diastereomer 2 (containing impurities) of the title compound. . Diastereomer 2 was purified again by preparative HPLC [water/acetonitrile containing 0.2% triethylamine, acetonitrile containing 0.2% triethylamine: 5% - 50% (0 min - 40 min)], and the diastereomer of the title compound was obtained. Stereomer 2 (94.6 mg) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00288
Figure pat00288

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00289
Figure pat00289

(공정 7-1) (Process 7-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-yl)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- (1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxo-14-(6, 7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (30 ㎎) 의 테트라하이드로푸란 (0.5 ㎖) 용액에 [(N-{[(9H-플루오렌-9-일)메톡시]카르보닐}글리실)아미노]메틸 아세테이트 (91.7 ㎎) 와 p-톨루엔술폰산 1수화물 (11.8 ㎎) 을 첨가하고, 실온에서 6 시간 교반하였다. 반응액에 N,N-디메틸포름아미드 (0.5 ㎖) 와 1,8-디아자비시클로[5.4.0]-7-운데센 (56 ㎕) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액에 10 mM 아세트산트리에틸암모늄 수용액을 첨가하고, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 불순물로서 원료를 포함하는 표제 화합물 (25.6 ㎎) 을 얻었다. [(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycyl to a solution of the compound (diastereomer 1) (30 mg) obtained in step 6 in tetrahydrofuran (0.5 ml) ) Amino] methyl acetate (91.7 mg) and p-toluenesulfonic acid monohydrate (11.8 mg) were added, and the mixture was stirred at room temperature for 6 hours. N,N-dimethylformamide (0.5 ml) and 1,8-diazabicyclo[5.4.0]-7-undecene (56 µl) were added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours. To the reaction solution was added an aqueous solution of 10 mM triethylammonium acetate, and purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain the title compound (25.6 mg) containing raw materials as impurities. .

MS(ESI)m/z : 1089(M＋H)＋.MS(ESI) m/z : 1089(M+H) + .

(공정 7-2) (Process 7-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- (1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxo-14-(6, 7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (84.6 ㎎) 을 사용하여, 상기 공정 7-1 과 동일한 수법으로 반응을 실시하고, 불순물로서 원료를 포함하는 표제 화합물 (70.9 ㎎) 을 얻었다. Using the compound (diastereomer 2) (84.6 mg) obtained in step 6 above, a reaction was carried out in the same manner as in step 7-1 to obtain the title compound (70.9 mg) containing raw materials as impurities.

MS(ESI)m/z : 1089(M＋H)＋.MS(ESI) m/z : 1089(M+H) + .

(공정 8-1) (Process 8-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) 디아스테레오머 1 Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}- 6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H -2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate) diastereomer 1

상기 공정 7-1 에서 얻어진 화합물 (25.6 ㎎) 에 트리에틸아민3불화수소산염 (2 ㎖) 을 첨가하고, 45 ℃ 에서 3 시간 교반하였다. 실온하, 반응액에 빙랭한 1 M 탄산수소트리에틸암모늄 용액 (10 ㎖) 과 트리에틸아민 (2 ㎖) 의 혼합액을 첨가하였다. 반응액을 감압 농축 후, C18 실리카 겔 칼럼 크로마토그래피 (10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴) 로 정제하고, 표제 화합물 (16.6 ㎎ : 공정 7-1 의 원료 유래의 불순물을 포함한다) 을 얻었다. Triethylamine hydrofluoric acid salt (2 ml) was added to the compound (25.6 mg) obtained in step 7-1, and the mixture was stirred at 45°C for 3 hours. To the reaction solution at room temperature, an ice-cooled mixture of 1 M triethylammonium hydrogen carbonate solution (10 ml) and triethylamine (2 ml) was added. The reaction solution was concentrated under reduced pressure and purified by C18 silica gel column chromatography (10 mM aqueous solution of triethylammonium acetate/acetonitrile) to obtain the title compound (16.6 mg: including impurities derived from the raw material in step 7-1). .

MS(ESI)m/z : 861(M＋H)＋.MS(ESI) m/z : 861(M+H) + .

(공정 8-2) (Process 8-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}- 6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H -2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7-2 에서 얻어진 화합물 (70.9 ㎎) 을 사용하여, 상기 공정 8-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (51.7 ㎎ : 공정 7-2 의 원료 유래의 불순물을 포함한다) 을 얻었다. Using the compound (70.9 mg) obtained in the above step 7-2, a reaction was carried out in the same manner as in the above step 8-1 to obtain the title compound (51.7 mg: containing impurities derived from the raw material of the step 7-2) got it

MS(ESI)m/z : 861(M＋H)＋.MS(ESI) m/z : 861(M+H) + .

(공정 9-1) (Process 9-1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-di Oxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7 -yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(약물 링커 2a : 디아스테레오머 1) (Drug linker 2a: diastereomer 1)

상기 공정 8-1 에서 얻어진 화합물 (16.6 ㎎) 의 N,N-디메틸포름아미드 (0.5 ㎖) 용액에 트리에틸아민 (6 ㎕) 과 후술하는 공정 11 에서 얻어진 화합물 (15.5 ㎎) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액에 벤질아민 (3 ㎕) 을 첨가하고, 실온에서 1 시간 교반하였다. 10 mM 아세트산트리에틸암모늄 수용액과 메탄올을 첨가하고, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 45 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (5.1 ㎎) 을 얻었다. To a solution of the compound (16.6 mg) obtained in step 8-1 above in N,N-dimethylformamide (0.5 ml) was added triethylamine (6 µl) and the compound (15.5 mg) obtained in step 11 described later (15.5 mg) were added to room temperature. was stirred for 3 hours. Benzylamine (3 µl) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. 10 mM triethylammonium acetate aqueous solution and methanol were added, and C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10 % - 45% (0 min - 30 min)] to obtain the title compound (5.1 mg).

Figure pat00290
Figure pat00290

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 9-2) (Process 9-2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-yl)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-di Oxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7 -yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(약물 링커 2b : 디아스테레오머 2) (Drug linker 2b: diastereomer 2)

상기 공정 8-2 에서 얻어진 화합물 (51.7 ㎎) 을 사용하여, 상기 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (33.7 ㎎) 을 얻었다. Using the compound (51.7 mg) obtained in the above step 8-2, the reaction was carried out in the same manner as in the above step 9-1, and then purified under the following [Purification conditions] to obtain the title compound (33.7 mg) .

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 50 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% - 50% (0 min - 30 minute)].

Figure pat00291
Figure pat00291

(공정 10) (Process 10)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닌 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanine

시판 (BACHEM) 되는 (2S)-2-[[2-[(2-아미노아세틸)아미노]아세틸]아미노]-3-페닐프로판산 (2.86 g) 의 N,N-디메틸포름아미드 (51.2 ㎖) 용액에 트리에틸아민 (2.56 ㎖) 과 시판 (Click Chemistry Tools) 되는 1-{[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]옥시}피롤리딘-2,5-디온 (3.69 g) 을 첨가하고, 실온에서 24 시간 교반하였다. 반응액에 시트르산 1수화물 (24.0 g) 의 물 (500 ㎖) 용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 아세트산에틸/아세토니트릴 혼합 용액에 용해시킨 후, 디이소프로필에테르로 석출시켜 여과 채취하고, 표제 화합물 (4.30 g) 을 얻었다. N,N-dimethylformamide (51.2 ml) of (2S)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-phenylpropanoic acid (2.86 g) commercially available (BACHEM) In solution, triethylamine (2.56 ml) and 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4- Oxobutanoyl]oxy}pyrrolidine-2,5-dione (3.69 g) was added, and the mixture was stirred at room temperature for 24 hours. A solution of citric acid monohydrate (24.0 g) in water (500 ml) was added to the reaction mixture, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. After dissolving the residue in a mixed solution of ethyl acetate/acetonitrile, the mixture was precipitated with diisopropyl ether and collected by filtration to obtain the title compound (4.30 g).

Figure pat00292
Figure pat00292

(공정 11) (Process 11)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라니네이트 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalaninate

상기 공정 10 에서 얻어진 화합물 (2.10 g) 의 N,N-디메틸포름아미드 (75.9 ㎖) 용액에 N-하이드록시숙신이미드 (961 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)카르보디이미드염산염 (1.60 g) 을 첨가하고, 질소 분위기하, 실온에서 21 시간 교반하였다. 반응액을 디클로로메탄으로 희석하고, 빙수로 3 회 세정한 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 톨루엔을 첨가하고, 재차 감압 농축하였다. 잔류물을 아세토니트릴에 용해하고, C18 실리카 겔 칼럼 크로마토그래피 [아세토니트릴 : 100 ％] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축 후, 잔류물에 디이소프로필에테르를 첨가하여 슬러리상으로 하였다. 얻어진 고체를 여과 채취하고, 표제 화합물 (2.59 g) 을 얻었다. N-hydroxysuccinimide (961 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carboxylate were added to a solution of the compound (2.10 g) obtained in step 10 in N,N-dimethylformamide (75.9 ml). Bodiimide hydrochloride (1.60 g) was added, and the mixture was stirred for 21 hours at room temperature under a nitrogen atmosphere. The reaction solution was diluted with dichloromethane, washed three times with ice water, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Toluene was added to the residue, and the mixture was again concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by C18 silica gel column chromatography [acetonitrile: 100%]. After concentrating the fraction containing the target product under reduced pressure, diisopropyl ether was added to the residue to form a slurry. The obtained solid was collected by filtration to obtain the title compound (2.59 g).

Figure pat00293
Figure pat00293

실시예 23 : 약물 링커 3 의 합성 Example 23: Synthesis of Drug Linker 3

[합성 스킴][Synthesis Scheme]

[화학식 150][Formula 150]

Figure pat00294
Figure pat00294

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2 ,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]glycinamide

(약물 링커 3) (drug linker 3)

실시예 8 공정 8-2 에서 얻어진 화합물 (4.8 ㎎) 의 N,N-디메틸포름아미드 (0.29 ㎖) 용액에, 트리에틸아민 (1.9 ㎕) 과 실시예 21 공정 3 에서 얻어진 화합물 (5.2 ㎎) 을 첨가하고, 질소 분위기하, 실온에서 1.5 시간 교반하였다. 벤질아민 (3.2 ㎕) 을 첨가하여 반응을 정지시킨 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (8.0 ㎎) 을 얻었다. To a solution of the compound (4.8 mg) obtained in Example 8 step 8-2 in N,N-dimethylformamide (0.29 ml), triethylamine (1.9 µl) and the compound obtained in Example 21 step 3 (5.2 mg) were added. This was added and stirred at room temperature in a nitrogen atmosphere for 1.5 hours. After stopping the reaction by adding benzylamine (3.2 µl), purification was performed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)]. Compound (8.0 mg) was obtained.

Figure pat00295
Figure pat00295

실시예 24 : 약물 링커 4 의 합성 Example 24: Synthesis of Drug Linker 4

[합성 스킴][Synthesis Scheme]

[화학식 151][Formula 151]

Figure pat00296
Figure pat00296

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-아미노-2-({2-[(글리실아미노)메톡시]에틸}아미노)-9H-푸린-9-일]-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-yl)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-amino-2-({2-[(glycylamino)methyl Toxy]ethyl}amino)-9H-purin-9-yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 - Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 9 공정 1-2 에서 얻어진 화합물 (17.6 ㎎) 을 사용하여, 실시예 22 공정 7-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (8.3 ㎎) 을 얻었다.Using the compound (17.6 mg) obtained in Example 9 Step 1-2, a reaction was carried out in the same manner as in Example 22 Step 7-1 to obtain the title compound (8.3 mg).

MS(ESI)m/z : 1103(M＋H)＋.MS(ESI)m/z: 1103(M+H)+.

(공정 2) (Process 2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-({2-[(글리실아미노)메톡시]에틸}아미노)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-({2-[(glycylamino)methyl Toxy]ethyl}amino)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3 ,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1 ,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 1 에서 얻어진 화합물 (10.7 ㎎) 을 사용하여, 실시예 22 공정 8-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (7.6 ㎎) 을 얻었다. Using the compound (10.7 mg) obtained in the above step 1, a reaction was carried out in the same manner as in Example 22 step 8-1 to obtain the title compound (7.6 mg).

MS(ESI)m/z : 875(M＋H)＋. MS(ESI) m/z : 875(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-{[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에톡시]메틸}글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-{[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy- 2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecin-7-yl]-9H-purin-2-yl}amino)ethoxy]methyl}glycinamide

(약물 링커 4) (Drug Linker 4)

상기 공정 2 에서 얻어진 화합물 (7.6 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시하였다. 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (7.6 ㎎) 을 트리에틸아민염으로서 얻었다. The reaction was carried out in the same manner as in Example 22 Step 9-1 using the compound (7.6 mg) obtained in Step 2 above. Purification was carried out under the following [Purification conditions] to obtain the title compound (7.6 mg) as a triethylamine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)].

Figure pat00297
Figure pat00297

실시예 25 : 당사슬 리모델링 항체 1 의 합성 Example 25: Synthesis of sugar chain remodeling antibody 1

개변 항HER2 항체 - [SG-(N3)2]2 의 합성 Modified anti-HER2 antibody - Synthesis of [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 152][Formula 152]

Figure pat00298
Figure pat00298

(공정 1) (Process 1)

(Fucα1,6)GlcNAc - 개변 항HER2 항체의 조제 Preparation of (Fucα1,6)GlcNAc-modified anti-HER2 antibody

참고예 1 에 따라서 조제한 개변 항HER2 항체의 인산 완충 생리 식염수 용액 (20 ㎖, 12.6 ㎎/㎖, pH 6.0) 에, 야생형 EndoS 의 인산 완충 생리 식염수 용액 (0.147 ㎖, 7.70 ㎎/㎖, pH 6.0) 을 첨가하고, 37 ℃ 에서 2 시간 15 분간 흔들었다. 반응의 진행 정도를 Experion 전기 영동 스테이션 (BIO-RAD 제조) 으로 확인하였다. 반응 종료 후, 이하의 방법에 따라서, 어피니티 크로마토그래피에 의한 정제와 하이드록시아파타이트 칼럼 크로마토그래피에 의한 정제를 실시하였다.To the modified anti-HER2 antibody prepared in Reference Example 1 in phosphate-buffered physiological saline solution (20 ml, 12.6 mg/ml, pH 6.0), a wild-type EndoS phosphate-buffered physiological saline solution (0.147 ml, 7.70 mg/ml, pH 6.0) was added. was added and shaken at 37°C for 2 hours and 15 minutes. The progress of the reaction was confirmed using an Experion electrophoresis station (manufactured by BIO-RAD). After completion of the reaction, purification by affinity chromatography and purification by hydroxyapatite column chromatography were performed according to the following methods.

(1) 어피니티 크로마토그래피에 의한 정제 (1) Purification by affinity chromatography

정제 장치 : AKTA avant 25 (GE 헬스 케어 제조) Refining device: AKTA avant 25 (manufactured by GE Healthcare)

칼럼 : HiTrap rProtein A FF (5 ㎖) (GE 헬스 케어 제조) Column: HiTrap rProtein A FF (5 ml) (manufactured by GE Healthcare)

유속 : 5 ㎖/분 (차지 시에는 1.25 ㎖/분) Flow rate: 5 ml/min (1.25 ml/min when charged)

상기에서 얻은 반응액을 2 회로 나누어 정제하였다. 칼럼으로의 결합 시에는, 반응액을 칼럼에 첨가하고, 결합 버퍼 [20 mM 인산 완충액 (pH 6.0)] 을 1.25 ㎖/분으로 2 CV 흘리고, 추가로 5 ㎖/분으로 5 CV 흘렸다. 중간 세정 시에는, 세정 용액 [20 mM 인산 완충액 (pH 7.0), 0.5 M 염화나트륨 용액] 을 15 CV 흘렸다. 용출 시에는, 용출 버퍼 (I㎜unoPure IgG Elution buffer, PIERCE 제조) 를 6 CV 흘렸다. 용출액을 1 M 트리스 완충액 (pH 9.0) 으로 즉시 중화하였다. 목적물을 포함하는 프랙션을 공통 조작 C 에 기재된 방법에 따라서, 5 mM 인산 완충액, 50 mM 2-모르폴리노에탄술폰산 (MES) 용액 (pH 6.8) 으로의 완충액 교환을 실시하였다. 얻어진 완충 용액의 항체 농도를 공통 조작 B 에 기재된 방법에 따라서 측정하고, 조정제된 표제 항체 용액 (26.57 ㎎/㎖, 9.0 ㎖) 을 얻었다. The reaction solution obtained above was divided into two times and purified. At the time of binding to the column, the reaction solution was added to the column, and 2 CVs of binding buffer [20 mM phosphate buffer (pH 6.0)] were flowed at 1.25 ml/min, and further 5 CVs were flowed at 5 ml/min. At the time of intermediate washing, 15 CV of washing solution [20 mM phosphate buffer (pH 7.0), 0.5 M sodium chloride solution] was flowed. During elution, 6 CV of elution buffer (ImmunoPure IgG Elution buffer, manufactured by PIERCE) was flowed. The eluate was immediately neutralized with 1 M Tris buffer (pH 9.0). The fraction containing the target object was subjected to buffer exchange with a 5 mM phosphate buffer, 50 mM 2-morpholinoethanesulfonic acid (MES) solution (pH 6.8) according to the method described in Common Operation C. The antibody concentration of the obtained buffer solution was measured according to the method described in common operation B, and the adjusted title antibody solution (26.57 mg/ml, 9.0 ml) was obtained.

(2) 하이드록시아파타이트 크로마토그래피에 의한 정제 (2) Purification by hydroxyapatite chromatography

정제 장치 : AKTA avant 25 (GE 헬스 케어 제조) Refining device: AKTA avant 25 (manufactured by GE Healthcare)

칼럼 : Bio-Scale Mini CHT Type I 카트리지 (5 ㎖) (BIO-RAD 제조) Column: Bio-Scale Mini CHT Type I cartridge (5 ml) (manufactured by BIO-RAD)

유속 : 5 ㎖/분 (차지 시에는 1.25 ㎖/분) Flow rate: 5 ml/min (1.25 ml/min when charged)

상기 (1) 에서 얻어진 용액을 칼럼에 첨가하고, A 액 [5 mM 인산 완충액, 50 mM MES 용액 (pH 6.8)] 을 1.25 ㎖/분으로 2 CV 흘리고, 추가로 5 ㎖/분으로 3 CV 흘렸다. 그 후, A 액과 B 액 [5 mM 인산 완충액, 50 mM MES 용액 (pH 6.8), 2 M 염화나트륨 용액] 을 사용하여, 용출하였다. 용출 조건은, A 액 : B 액 = 100 : 0 ∼ 0 : 100 (5 CV) 이다. 또한 세정 용액 [500 mM 인산 완충액 (pH 6.5)] 을 5 CV 흘렸다. 목적물을 포함하는 프랙션을 공통 조작 C 에 기재된 방법에 따라서, 20 mM 인산 완충액 (pH 6.0) 으로의 완충액 교환을 실시하였다. 얻어진 완충 용액의 항체 농도를 공통 조작 B 에 기재된 방법에 따라서 측정하고, 표제 항체 용액 (17.29 ㎎/㎖, 약 13 ㎖) 을 얻었다.The solution obtained in (1) above was added to the column, and 2 CV of Solution A [5 mM phosphate buffer, 50 mM MES solution (pH 6.8)] was flowed at 1.25 ml/min, and further 3 CV was flowed at 5 ml/min. . Thereafter, elution was performed using Liquid A and Liquid B [5 mM phosphate buffer, 50 mM MES solution (pH 6.8), 2 M sodium chloride solution]. Elution conditions are liquid A : liquid B = 100:0 to 0:100 (5 CV). Further, 5 CV of washing solution [500 mM phosphate buffer (pH 6.5)] was flowed. The fraction containing the target object was subjected to buffer exchange with a 20 mM phosphate buffer (pH 6.0) according to the method described in common operation C. The antibody concentration of the obtained buffer solution was measured according to the method described in common operation B, and the title antibody solution (17.29 mg/ml, about 13 ml) was obtained.

(공정 2) (Process 2)

개변 항HER2 항체 - [SG-(N3)2]2 의 조제 Modified anti-HER2 antibody - Preparation of [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (17.29 ㎎/㎖, 13 ㎖, pH 6.0) 에 [N3-PEG(3)]2-SG(10)Ox (WO2018/003983 의 화합물 1-10) (52 ㎎) 의 20 mM 인산 완충 용액 (pH 6.0) (3.0 ㎖ ＋ 씻어내기 1.0 ㎖) 및 EndoS (D233Q/Q303L) 의 인산 완충 생리 식염수 용액 (0.698 ㎖, 5.8 ㎎/㎖, pH 6.0) 을 첨가하고, 30 ℃ 에서 4 시간 흔들었다. 반응액을 -80 ℃ 에서 15 시간 보존한 후, 30 ℃ 에서 해동하고, [N3-PEG(3)]2-SG(10)Ox (7.4 ㎎) 및 EndoS (D233Q/Q303L) 의 인산 완충 생리 식염수 용액 (0.155 ㎖, 5.8 ㎎/㎖, pH 6.0) 을 추가하여 30 ℃ 에서 2 시간 흔들었다. 반응의 진행 정도를 Experion 전기 영동 스테이션 (BIO-RAD 제조) 으로 확인하였다. 반응 종료 후, 상기 공정 1 과 동일하게 어피니티 크로마토그래피에 의한 정제와 하이드록시아파타이트 크로마토그래피에 의한 정제를 실시하였다. 목적물을 포함하는 프랙션 (합계 7 개) 을 전의 4 개와 후의 3 개로 나누고, 각각을 공통 조작 C 에 기재된 방법에 따라서, 인산 완충 생리 식염수 (pH 6.0) 로의 완충액 교환을 실시하였다. 얻어진 완충 용액의 항체 농도를 공통 조작 B 에 기재된 방법에 따라서 측정하고, 표제 항체 용액 (전의 4 개 : 14.99 ㎎/㎖, 10 ㎖) 과 표제 항체 용액 (후의 3 개 : 10.97 ㎎/㎖, 6.2 ㎖) 을 각각 얻었다.[N 3 -PEG(3)] 2 -SG(10)Ox (Compound 1-10 of WO2018/003983 ) (52 mg) in 20 mM phosphate buffer solution (pH 6.0) (3.0 ml + rinse 1.0 ml) and EndoS (D233Q/Q303L) in phosphate buffered saline solution (0.698 ml, 5.8 mg/ml, pH 6.0) It was added and shaken at 30 degreeC for 4 hours. The reaction solution was stored at -80°C for 15 hours, then thawed at 30°C, and [N 3 -PEG(3)] 2 -SG(10)Ox (7.4 mg) and EndoS (D233Q/Q303L) phosphate buffer physiology A saline solution (0.155 ml, 5.8 mg/ml, pH 6.0) was added, and the mixture was shaken at 30°C for 2 hours. The progress of the reaction was confirmed using an Experion electrophoresis station (manufactured by BIO-RAD). After completion of the reaction, purification by affinity chromatography and purification by hydroxyapatite chromatography were performed in the same manner as in Step 1 above. Fractions containing the target object (7 pieces in total) were divided into 4 before and 3 after, and each was buffer-exchanged with phosphate buffered saline (pH 6.0) according to the method described in common operation C. The antibody concentration of the obtained buffer solution was measured according to the method described in common operation B, and the title antibody solution (4 former: 14.99 mg/mL, 10 mL) and the title antibody solution (later 3: 10.97 mg/mL, 6.2 mL) were obtained. ) were obtained, respectively.

실시예 26 : 당사슬 리모델링 항체 2 의 합성 Example 26: Synthesis of sugar chain remodeling antibody 2

개변 항LPS 항체 - [SG-(N3)2]2 의 조제 Modified anti-LPS antibody - Preparation of [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 153][Formula 153]

Figure pat00299
Figure pat00299

(공정 1) (Process 1)

(Fucα1,6)GlcNAc - 개변 항LPS 항체의 조제 Preparation of (Fucα1,6)GlcNAc-modified anti-LPS antibody

참고예 2 에 따라서 조제한 개변 항LPS 항체의 인산 완충 생리 식염수 용액 (8.5 ㎖, 10.96 ㎎/㎖, pH 6.0) 을 사용하여, 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (11.70 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.Using the phosphate-buffered physiological saline solution (8.5 mL, 10.96 mg/mL, pH 6.0) of the modified anti-LPS antibody prepared according to Reference Example 2, the same operation as in Example 25 Step 1 was performed, and the 20 mM phosphate of the title antibody A buffer solution (11.70 mg/ml, 7.5 ml, pH 6.0) was obtained.

(공정 2) (Process 2)

개변 항LPS 항체 - [SG-(N3)2]2 의 조제 Modified anti-LPS antibody - Preparation of [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (11.70 ㎎/㎖, 7.5 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (20.3 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.55 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (11.70 mg/mL, 7.5 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (20.3 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (10.55 mg/ml, 7.5 ml, pH 6.0) of the title antibody.

실시예 27 : 항체 약물 콘쥬게이트 1 의 합성 (항HER2 항체 - CDN 콘쥬게이트 1 의 합성) Example 27: Synthesis of Antibody Drug Conjugate 1 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 1)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.97 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 3 의 디메틸술폭시드 용액 (10 mM, 0.0907 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.159 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS (10 mM Acetate Buffer, 5 ％ Sorbitol, pH 5.5) 용액 (3.5 ㎖) 을 얻었다.A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.97 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 3 dimethylsulfoxide solution (10 mM, 0.0907 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.159 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS (10 mM Acetate Buffer, 5% Sorbitol, pH 5.5) solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.97 ㎎/㎖ Antibody concentration: 0.97 mg/ml

항체 수량 (收量) : 3.41 ㎎ (62 ％) Antibody Quantity: 3.41 mg (62%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 28 : 항체 약물 콘쥬게이트 2 의 합성 (항HER2 항체 - CDN 콘쥬게이트 2 의 합성) Example 28: Synthesis of Antibody Drug Conjugate 2 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 2)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.97 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 4 의 디메틸술폭시드 용액 (10 mM, 0.0907 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.159 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.97 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 4 dimethylsulfoxide solution (10 mM, 0.0907 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.159 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.08 ㎎/㎖ Antibody concentration: 1.08 mg/ml

항체 수량 : 3.78 ㎎ (69 ％) Antibody quantity: 3.78 mg (69%)

약물 평균 결합수 : 3.2Average number of drug bonds: 3.2

실시예 29 : 항체 약물 콘쥬게이트 3 의 합성 (항HER2 항체 - CDN 콘쥬게이트 3 의 합성) Example 29: Synthesis of Antibody Drug Conjugate 3 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 3)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.97 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2a 의 디메틸술폭시드 용액 (10 mM, 0.0907 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.159 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 47 시간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.97 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2a dimethylsulfoxide solution (10 mM, 0.0907 ml, 24 equivalents per antibody molecule) and propylene glycol (0.159 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 47 hours. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.91 ㎎/㎖ Antibody concentration: 0.91 mg/ml

항체 수량 : 3.17 ㎎ (58 ％) Antibody quantity: 3.17 mg (58%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 30 : 항체 약물 콘쥬게이트 4 의 합성 (항LPS 항체 - CDN 콘쥬게이트 1 의 합성) Example 30: Synthesis of Antibody Drug Conjugate 4 (Synthesis of Anti-LPS Antibody-CDN Conjugate 1)

당사슬 리모델링 항체 2 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.55 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 3 의 디메틸술폭시드 용액 (10 mM, 0.174 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.326 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (6.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 2 in phosphate buffered saline (pH 6.0) (10.55 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 3 dimethylsulfoxide solution (10 mM, 0.174 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.326 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (6.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.11 ㎎/㎖ Antibody concentration: 1.11 mg/ml

항체 수량 : 7.23 ㎎ (69 ％) Antibody quantity: 7.23 mg (69%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 31 : CDN21 의 합성 Example 31: Synthesis of CDN21

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) )-14-(7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd]inden-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-dione

[화학식 154][Formula 154]

Figure pat00300
Figure pat00300

[합성 스킴][Synthesis Scheme]

[화학식 155][Formula 155]

Figure pat00301
Figure pat00301

(공정 1) (Process 1)

4-(벤질옥시)-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(4-하이드록시부타-1-인-1-일)-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl} -5-(4-hydroxybuta-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine

실시예 20 공정 2 에서 얻어진 화합물 (3.37 g) 과 3-부틴-1-올 (1.72 ㎖) 을 사용하여, 반응 온도를 실온으로 하여, 실시예 1 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.74 g) 을 얻었다. Example 20 Using the compound (3.37 g) obtained in Step 2 and 3-butyn-1-ol (1.72 ml), the reaction temperature was room temperature, and the reaction was carried out in the same manner as in Example 1 Step 2. A compound (2.74 g) was obtained.

Figure pat00302
Figure pat00302

(공정 2) (Process 2)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(4-하이드록시부틸)-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-온 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(4-hydride Roxybutyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

상기 공정 1 에서 얻어진 화합물 (2.74 g) 을 사용하여, 반응 용매를 메탄올 (30 ㎖) - 테트라하이드로푸란 (30 ㎖) 혼합 용매로 하고, 실시예 19 공정 7 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.21 g) 을 얻었다.Using the compound (2.74 g) obtained in the above step 1, the reaction was carried out in the same manner as in Example 19 step 7, using a mixed solvent of methanol (30 ml) and tetrahydrofuran (30 ml) as the reaction solvent, A compound (2.21 g) was obtained.

Figure pat00303
Figure pat00303

(공정 3) (Process 3)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-7,8,9, 10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd]indene

상기 공정 2 에서 얻어진 화합물 (1.89 g) 을 사용하여, 실시예 20 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.40 g) 을 얻었다. Using the compound (1.89 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 20 step 5 to obtain the title compound (1.40 g).

Figure pat00304
Figure pat00304

(공정 4) (Process 4)

2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴 2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-7,8, 9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd]indene

상기 공정 3 에서 얻어진 화합물 (1.61 g) 을 사용하여, 실시예 1 의 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.95 g) 을 얻었다. Using the compound (1.61 g) obtained in the above step 3, a reaction was carried out in the same manner as in step 5 of Example 1 to obtain the title compound (1.95 g).

Figure pat00305
Figure pat00305

(공정 5) (Process 5)

2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴 2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacyclo octa[1,2,3-cd]indene

상기 공정 4 에서 얻어진 화합물 (1.95 g) 을 사용하여, 실시예 5 공정 4 와 동일하게 조작하고, 표제 화합물 (2.20 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다.Using the compound (1.95 g) obtained in step 4 above, the same procedure as in Example 5 step 4 was used to obtain the title compound (2.20 g) as a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 6:4). .

Figure pat00306
Figure pat00306

(공정 6) (Process 6)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-비스(술파닐)-14-(7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy } -2,10-bis(sulfanyl)-14-(7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd ]inden-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 5 에서 얻어진 화합물 (1.18 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-O-[tert-부틸(디메틸)실릴]-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 시판 (Cool Pharm) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.49 g) 을 사용하여, 실시예 1 공정 8, 공정 9, 및 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 이 혼합물을 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 60 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물의 디아스테레오머 1 (50 ㎎) 과 디아스테레오머 2 (34 ㎎) 를 얻었다. Using the compound (1.18 g) obtained in step 5 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[ Hydroxy (oxo)-λ 5 -phosphanyl] -β-D-ribofuranosyl} -7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta An acetonitrile solution of [1,2,3-cd]indene was obtained. This acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]- Example 1 Steps 8, 9, and 10 using 2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.49 g) The reaction was carried out in the same manner to obtain the title compound as a mixture of diastereomers on a phosphorus atom. This mixture was purified by HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 25% - 60% (0 min - 35 min)] to obtain diastereomer 1 (50 mg) of the title compound and diastereomer Mercury 2 (34 mg) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 973(M＋H)＋. MS(ESI) m/z : 973(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 973(M＋H)＋.MS(ESI) m/z : 973(M+H) + .

(공정 7-1) (Process 7-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd]inden-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (50 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 1) (50 mg) obtained in Step 6 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (33 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (33 mg).

Figure pat00307
Figure pat00307

(공정 7-2) (Process 7-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(7,8,9,10-테트라하이드로-2H-6-옥사-2,3,5-트리아자시클로옥타[1,2,3-cd]인덴-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(7,8,9,10-tetrahydro-2H-6-oxa-2,3,5-triazacycloocta[1,2,3-cd]inden-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (34 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 2) (34 mg) obtained in Step 6 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21 mg).

Figure pat00308
Figure pat00308

실시예 32 : CDN22 의 합성 Example 32: Synthesis of CDN22

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-14-[(7S)-7-메틸-8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일]-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-14-[(7S)-7 -Methyl-8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl]-2,10-bis(sulfanyl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-dione

[화학식 156][Formula 156]

Figure pat00309
Figure pat00309

[합성 스킴][Synthesis Scheme]

[화학식 157][Formula 157]

Figure pat00310
Figure pat00310

(공정 1) (Process 1)

4-(벤질옥시)-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-[(3R)-3-하이드록시부타-1-인-1-일]-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl} -5-[(3R)-3-hydroxybuta-1-yn-1-yl]-7H-pyrrolo[2,3-d]pyrimidine

실시예 20 공정 2 에서 얻어진 화합물 (2.54 g) 과 (R)-(＋)-3-부틴-2-올 (1.36 ㎖) 을 사용하여, 반응 온도를 실온으로 하고, 실시예 1 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.85 g) 을 얻었다. Using the compound (2.54 g) obtained in Example 20 Step 2 and (R)-(+)-3-butyn-2-ol (1.36 mL), the reaction temperature was set to room temperature, and the same reaction as in Example 1 Step 2 was performed. The reaction was carried out by the method to obtain the title compound (1.85 g).

Figure pat00311
Figure pat00311

(공정 2) (Process 2)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-[(3R)-3-하이드록시부틸]-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-온 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-[(3R) -3-hydroxybutyl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

상기 공정 1 에서 얻어진 화합물 (1.85 g) 을 사용하여, 반응 용매를 메탄올 (15 ㎖) - 테트라하이드로푸란 (15 ㎖) 혼합 용매로 하고, 실시예 19 공정 7 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.34 g) 을 얻었다. Using the compound (1.85 g) obtained in the above step 1, the reaction was carried out in the same manner as in Example 19 step 7, using a mixed solvent of methanol (15 ml) and tetrahydrofuran (15 ml) as the reaction solvent, A compound (1.34 g) was obtained.

Figure pat00312
Figure pat00312

(공정 3) (Process 3)

(7S)-2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-7-메틸-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 (7S)-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-7- Methyl-2,7,8,9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulene

상기 공정 2 에서 얻어진 화합물 (1.34 g) 을 사용하여, 실시예 20 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (0.70 g) 을 얻었다. Using the compound (1.34 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 20 step 5 to obtain the title compound (0.70 g).

Figure pat00313
Figure pat00313

(공정 4) (Process 4)

(7S)-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-7-메틸-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 (7S)-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}- 7-methyl-2,7,8,9-tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulene

상기 공정 3 에서 얻어진 화합물 (0.70 g) 을 사용하여, 실시예 1 의 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (0.82 g) 을 얻었다. Using the compound (0.70 g) obtained in the above step 3, a reaction was carried out in the same manner as in step 5 of Example 1 to obtain the title compound (0.82 g).

Figure pat00314
Figure pat00314

(공정 5) (Process 5)

(7S)-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-7-메틸-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌 (7S)-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-7-methyl-2,7,8,9-tetrahydro-6-oxa-2,3 ,5-triazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (0.82 g) 을 사용하여, 실시예 5 의 공정 4 와 동일하게 조작하고, 표제 화합물 (0.85 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. Using the compound (0.82 g) obtained in step 4 above, the same procedure as in step 4 of Example 5 was used to obtain the title compound (0.85 g) as a phosphorus atom-phase diastereomer mixture (diastereomer ratio = 6:4). got it

Figure pat00315
Figure pat00315

(공정 6) (Process 6)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-14-[(7S)-7-메틸-8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일]-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy }-14-[(7S)-7-methyl-8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl]-2,10 -bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 5 에서 얻어진 화합물 (0.85 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, (7S)-2-{2-O-[tert-부틸(디메틸)실릴]-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-7-메틸-2,7,8,9-테트라하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 시판 (Cool Pharm) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.12 g) 을 사용하여, 실시예 1 공정 8, 공정 9, 및 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 이 혼합물을 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 60 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물의 디아스테레오머 1 (95 ㎎) 과 디아스테레오머 2 (44 ㎎) 를 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). Using the compound (0.85 g) obtained in step 5 above, reaction was carried out in the same manner as in step 7 of Example 1, (7S)-2-{2-O-[tert-butyl(dimethyl)silyl]-3 -O-[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-7-methyl-2,7,8,9-tetrahydro-6-oxa-2,3; An acetonitrile solution of 5-triazabenzo[cd]azulene was obtained. This acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]- Example 1 Steps 8, 9, and 10 using 2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.12 g) The reaction was carried out in the same manner to obtain the title compound as a mixture of diastereomers on a phosphorus atom. This mixture was purified by HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 25% - 60% (0 min - 35 min)] to obtain diastereomer 1 (95 mg) of the title compound and diastereomer Mercury 2 (44 mg) was obtained (retention time by HPLC: diastereomer 1 > 2).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 973(M＋H)＋. MS(ESI) m/z : 973(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 973(M＋H)＋.MS(ESI) m/z : 973(M+H) + .

(공정 7-1) (Process 7-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-14-[(7S)-7-메틸-8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일]-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-14-[(7S) -7-methyl-8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl]-2,10-dioxoooctahydro-2H; 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-2; 10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (95 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 1) (95 mg) obtained in Step 6 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (57 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as described in Example 1, step 11 [Conversion to sodium salt] to obtain the title compound (57 mg).

Figure pat00316
Figure pat00316

(공정 7-2) (Process 7-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-14-[(7S)-7-메틸-8,9-디하이드로-6-옥사-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일]-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-14-[(7S) -7-methyl-8,9-dihydro-6-oxa-2,3,5-triazabenzo[cd]azulen-2(7H)-yl]-2,10-dioxoooctahydro-2H; 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-2; 10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (44 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 2) (44 mg) obtained in Step 6 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (30 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (30 mg).

Figure pat00317
Figure pat00317

실시예 33 : CDN23 의 합성 Example 33: Synthesis of CDN23

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-thia-2; 3,5-triazabenzo[cd]azulen-2(7H)-yl)-15,16-dihydroxy-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 158][Formula 158]

Figure pat00318
Figure pat00318

[합성 스킴][Synthesis Scheme]

[화학식 159][Formula 159]

Figure pat00319
Figure pat00319

(공정1) (Process 1)

4-(벤질옥시)-5-{3-[비스(4-메톡시페닐)(페닐)메톡시]프로파-1-인-1-일}-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-5-{3-[bis(4-methoxyphenyl)(phenyl)methoxy]prop-1-yn-1-yl}-7-{2-O-[tert-butyl (dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-7H-pyrrolo[2,3-d]pyrimidine

실시예 20 공정 3 에서 얻어진 화합물 (4.17 g) 을 사용하여, 반응 용매를 디클로로메탄 (40 ㎖) - 피리딘 (40 ㎖) 혼합 용매로 하고, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.70 g) 을 얻었다. Using the compound (4.17 g) obtained in Example 20 Step 3, the reaction was carried out in the same manner as in Example 11 Step 1 using a dichloromethane (40 mL)-pyridine (40 mL) mixed solvent as the reaction solvent, The title compound (5.70 g) was obtained.

Figure pat00320
Figure pat00320

(공정 2) (Process 2)

5-{3-[비스(4-메톡시페닐)(페닐)메톡시]프로필}-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-온 5-{3-[bis(4-methoxyphenyl)(phenyl)methoxy]propyl}-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert -Butylsilylidene)-β-D-ribofuranosyl}-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

상기 공정 1 에서 얻어진 화합물 (5.70 g) 의 메탄올 (100 ㎖) - 테트라하이드로푸란 (50 ㎖) 혼합 용액에, 포름산암모늄 (3.71 g) 과 10 ％ 팔라듐탄소 (AD) wet (2g) 를 첨가하고, 실온에서 3 시간 교반하였다. 촉매를 여과 제거한 후, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (4.56 g) 을 얻었다. Ammonium formate (3.71 g) and 10% palladium carbon (AD) wet (2 g) were added to a methanol (100 ml)-tetrahydrofuran (50 ml) mixed solution of the compound (5.70 g) obtained in step 1 above, It was stirred at room temperature for 3 hours. After filtering off the catalyst, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (4.56 g).

Figure pat00321
Figure pat00321

(공정 3) (Process 3)

5-{3-[비스(4-메톡시페닐)(페닐)메톡시]프로필}-7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-티온 5-{3-[bis(4-methoxyphenyl)(phenyl)methoxy]propyl}-7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert -Butylsilylidene)-β-D-ribofuranosyl}-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-thione

상기 공정 2 에서 얻어진 화합물 (1.01 g) 의 디클로로메탄 (10 ㎖) 용액에, 피리딘 (0.461 ㎖) 을 첨가하고, 빙랭하에서 트리플루오로메탄술폰산 무수물 (0.385 ㎖) 을 적하하고 30 분간 교반하였다. 동 온도에서 반응액에 1 황화수소나트륨 n 수화물 (2.54 g) 의 N,N-디메틸포르미아미드 (25 ㎖) 현탁액을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 아세트산에틸을 첨가하고, 셀라이트로 여과한 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (0.51 g) 을 얻었다. To a solution of the compound (1.01 g) obtained in step 2 above in dichloromethane (10 ml), pyridine (0.461 ml) was added, and trifluoromethanesulfonic anhydride (0.385 ml) was added dropwise under ice-cooling, followed by stirring for 30 minutes. A suspension of sodium hydrogen sulfide n hydrate (2.54 g) in N,N-dimethylformiamide (25 ml) was added to the reaction solution at the same temperature, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous solution of sodium hydrogen carbonate and ethyl acetate were added to the reaction solution, and after filtration through celite, extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (0.51 g).

Figure pat00322
Figure pat00322

(공정 4) (Process 4)

7-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-5-(3-하이드록시프로필)-3,7-디하이드로-4H-피롤로[2,3-d]피리미딘-4-티온 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(3-hydride Roxypropyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-thione

상기 공정 3 에서 얻어진 화합물 (2.79 g) 의 디클로로메탄 (80 ㎖) 용액에, 증류수 (4 ㎖) 를 첨가하고, 빙랭하에서 디클로로아세트산 (1.28 ㎖) 을 적하하고 30 분간 교반하였다. 동 온도에서 반응액에 피리딘 (2.50 ㎖) 을 첨가한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (0.96 g) 을 얻었다. To a dichloromethane (80 ml) solution of the compound (2.79 g) obtained in step 3 above, distilled water (4 ml) was added, dichloroacetic acid (1.28 ml) was added dropwise under ice-cooling, and the mixture was stirred for 30 minutes. After adding pyridine (2.50 ml) to the reaction solution at the same temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (0.96 g).

Figure pat00323
Figure pat00323

(공정 5) (Process 5)

2-{2-O-[tert-부틸(디메틸)실릴]-3,5-O-(디-tert-부틸실릴리덴)-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-2,7,8, 9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (0.35 g) 을 사용하여, 실시예 20 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (0.25 g) 을 얻었다. Using the compound (0.35 g) obtained in the above step 4, a reaction was carried out in the same manner as in Example 20 step 5 to obtain the title compound (0.25 g).

Figure pat00324
Figure pat00324

(공정 6) (Process 6)

2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-2,7, 8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 5 에서 얻어진 화합물 (0.41 g) 을 사용하여, 실시예 1 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (0.46 g) 을 얻었다.Using the compound (0.41 g) obtained in step 5 above, reaction was carried out in the same manner as in step 5 of Example 1 to obtain the title compound (0.46 g).

Figure pat00325
Figure pat00325

(공정 7) (Process 7)

2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-β-D-리보퓨라노실)-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd ]azulene

상기 공정 6 에서 얻어진 화합물 (0.46 g) 을 사용하여, 실시예 5 공정 4 와 동일하게 조작하고, 표제 화합물 (0.48 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다. Using the compound (0.46 g) obtained in Step 6 above, the same procedure as in Example 5 Step 4 was used to obtain the title compound (0.48 g) as a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 6:4). .

Figure pat00326
Figure pat00326

(공정 8) (Process 8)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoe Toxy)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-2-oxo-2-sulfanyl-10 -sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 7 에서 얻어진 화합물 (0.48 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-O-[tert-부틸(디메틸)실릴]-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 시판 (Cool Pharm) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (0.61 g) 을 사용하여, 실시예 1 공정 8, 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 이 혼합물을 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 40 ％ - 90 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물의 디아스테레오머 1 (40 ㎎) 과 디아스테레오머 2 (18 ㎎) 를 각각 얻었다.Using the compound (0.48 g) obtained in step 7 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[ Hydroxy (oxo)-λ 5 -phosphanyl] -β-D-ribofuranosyl} -2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo [cd] A solution of azulene in acetonitrile was obtained. This acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]- 2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (0.61 g) was used and reacted in the same manner as in Example 1 Steps 8 and 9 was carried out to obtain the title compound as a mixture of diastereomers on the phosphorus atom. The mixture was purified by HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 40% - 90% (0 min - 35 min)] to obtain diastereomer 1 (40 mg) of the title compound and diastereomer Mer 2 (18 mg) was obtained respectively.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1132(M＋H)＋. MS(ESI)m/z: 1132(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1132(M＋H)＋.MS(ESI)m/z: 1132(M+H) + .

(공정 9-1) (Process 9-1)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy } -14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-2,10-bis(sulfanyl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -2,10-dione

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 1) (40 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 60 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물 (35 ㎎) 을 얻었다.Using the compound (diastereomer 1) (40 mg) obtained in step 8 above, reaction was carried out in the same manner as in step 10 of Example 1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile : 30% - 60% (0 min - 35 min)] to obtain the title compound (35 mg).

MS(ESI)m/z : 975(M＋H)＋.MS(ESI) m/z : 975(M+H) + .

(공정 9-2) (Process 9-2)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy } -14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-2,10-bis(sulfanyl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -2,10-dione

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 2) (18 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 50 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물 (15 ㎎) 을 얻었다. Using the compound (diastereomer 2) (18 mg) obtained in step 8 above, reaction was carried out in the same manner as in step 10 of Example 1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile : 25% - 50% (0 min - 35 min)] to obtain the title compound (15 mg).

MS(ESI)m/z : 975(M＋H)＋.MS(ESI) m/z : 975(M+H) + .

(공정 10-1) (Process 10-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-thia- 2,3,5-triazabenzo[cd]azulene-2(7H)-yl)-15,16-dihydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8- Methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 9-1 에서 얻어진 화합물 (35 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (35 mg) obtained in Step 9-1 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/acetonitrile = 5 : 1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (18 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (18 mg).

Figure pat00327
Figure pat00327

(공정 10-2) (Process 10-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15,16-디하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-14-(8,9-dihydro-6-thia- 2,3,5-triazabenzo[cd]azulene-2(7H)-yl)-15,16-dihydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8- Methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 9-2 에서 얻어진 화합물 (15 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (15 mg) obtained in Step 9-2 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/acetonitrile = 5 : 1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (8.1 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (8.1 mg).

Figure pat00328
Figure pat00328

실시예 34 : CDN24 의 합성 Example 34: Synthesis of CDN24

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]피리미딘-2,4(1H,3H)-디온1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis(sulfanyl)-14-(6 ,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]pyrimidine-2,4(1H,3H ) - Dion

[화학식 160][Formula 160]

Figure pat00329
Figure pat00329

[합성 스킴][Synthesis Scheme]

[화학식 161][Formula 161]

Figure pat00330
Figure pat00330

(공정 1) (Process 1)

1-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]피리미딘-2,4(1H,3H)-디온 1-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-2,10- bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H; 12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl] Pyrimidine-2,4(1H,3H)-dione

실시예 1 공정 7 의 반응을 이하의 스케일로 실시하였다 (원료 : 1.01 g). 얻어진 화합물의 아세토니트릴 용액과 시판 (Angene) 되는 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}우리딘 (1.03 g) 을 사용하여, 실시예 1 의 공정 8, 공정 9, 및 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. 이 혼합물을 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 60 ％ (0 분 - 35 분)] 로 정제하고, 표제 화합물의 디아스테레오머 1 (50 ㎎) 과 디아스테레오머 2 (23 ㎎) 를 얻었다. The reaction in step 7 of Example 1 was carried out on the following scale (raw material: 1.01 g). Acetonitrile solution of the obtained compound and commercially available (Angene) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'- Using O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}uridine (1.03 g), steps 8, 9, and 10 of Example 1 were used. The reaction was carried out by this method to obtain the title compound as a mixture of diastereomers on a phosphorus atom. This mixture was purified by HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 25% - 60% (0 min - 35 min)] to obtain diastereomer 1 (50 mg) of the title compound and diastereomer Mercury 2 (23 mg) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 935(M＋H)＋. MS(ESI) m/z : 935(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 935(M＋H)＋.MS(ESI) m/z : 935(M+H) + .

(공정 2-1) (Step 2-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2,4-디옥소-3,4-디하이드로 피리미딘-1(2H)-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-15,16 -Dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 1) (50 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 1) (50 mg) obtained in Step 1 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (30 ㎎) 을 얻었다. The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (30 mg).

Figure pat00331
Figure pat00331

(공정 2-2) (Process 2-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2,4-디옥소-3,4-디하이드로 피리미딘-1(2H)-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-15,16 -Dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 2) (23 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴 = 5 : 1] 로 정제하고, 표제 화합물의 트리에틸아민염을 얻었다. After reacting in the same manner as in Example 1 Step 11 using the compound (Diastereomer 2) (23 mg) obtained in Step 1 above, Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/ acetonitrile = 5:1] to obtain the triethylamine salt of the title compound.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (11 ㎎) 을 얻었다. The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (11 mg).

Figure pat00332
Figure pat00332

실시예 35 : CDN25 의 합성 Example 35: Synthesis of CDN25

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-(6-옥소-1,6-디하이드로-9H-푸린-9-일)-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2 ,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2 ,10-dione

[화학식 162][Formula 162]

Figure pat00333
Figure pat00333

[합성 스킴][Synthesis Scheme]

[화학식 163][Formula 163]

Figure pat00334
Figure pat00334

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-7-(6-옥소-1,6-디하이드로-9H-푸린-9-일)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2, 10-dioxo-7-(6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3, 6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 13 공정 4 에서 얻어진 화합물 (313 ㎎) 의 테트라하이드로푸란 (5.0 ㎖) 용액에, N-[(E)-(피리딘-2-일)메틸리덴]하이드록실아민 (337 ㎎) 과 N,N,N',N'-테트라메틸구아니딘 (0.346 ㎖) 을 첨가하고, 실온에서 1 일간 교반하였다. 반응액에 메탄올 (5.0 ㎖) 과 28 ％ 암모니아수 (5.0 ㎖) 를 첨가하고, 50 ℃ 에서 5 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (106 ㎎ : 불순물 함유) 과 디아스테레오머 2 (105 ㎎ : 불순물 함유) 를 얻었다. Example 13 To a solution of the compound (313 mg) obtained in step 4 in tetrahydrofuran (5.0 ml), N-[(E)-(pyridin-2-yl)methylidene]hydroxylamine (337 mg) and N, N,N',N'-tetramethylguanidine (0.346 ml) was added, and the mixture was stirred at room temperature for 1 day. Methanol (5.0 ml) and 28% aqueous ammonia (5.0 ml) were added to the reaction solution, and the mixture was stirred at 50°C for 5 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (106 mg: containing impurities) and diastereomer of the title compound. 2 (105 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 959(M＋H)＋. MS(ESI) m/z : 959(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 959(M＋H)＋.MS(ESI) m/z : 959(M+H) + .

(공정 2-1) (Step 2-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-7-(6-옥소-1,6-디하이드로-9H-푸린-9-일)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5, 10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-7-(6-oxo-1,6-dihydro-9H -Purin-9-yl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H, 10H,12H-5,8-methano-2λ 5 , 10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2; 10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 1) (106 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (106 mg: containing impurities) obtained in Step 1 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 7 ％ - 50 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 7% - 50% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (43.4 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (43.4 mg).

Figure pat00335
Figure pat00335

(공정 2-2) (Process 2-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-7-(6-옥소-1,6-디하이드로-9H-푸린-9-일)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-7-(6-oxo-1,6-dihydro-9H -Purin-9-yl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H, 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-2; 10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 1 에서 얻어진 화합물 (디아스테레오머 2) (105 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (105 mg: containing impurities) obtained in Step 1 above, the reaction was carried out in the same manner as in Step 11 of Example 1, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 7 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 7% - 45% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21.5 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21.5 mg).

Figure pat00336
Figure pat00336

실시예 36 : CDN26 의 합성 Example 36: Synthesis of CDN26

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(3-하이드록시프로필)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(3-hydroxypropyl)-6-oxo-1,6-dihydro- 9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 164][Formula 164]

Figure pat00337
Figure pat00337

[합성 스킴][Synthesis Scheme]

[화학식 165][Formula 165]

Figure pat00338
Figure pat00338

(공정 1) (Process 1)

1-[3-(벤조일옥시)프로필]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]이노신1-[3-(benzoyloxy)propyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine

3-브로모프로판-1-올 (1.14 ㎖) 의 테트라하이드로푸란 (25 ㎖) 용액에, 트리에틸아민 (1.83 ㎖) 과 염화벤조일 (1.43 ㎖) 을 첨가하고, 실온에서 6 시간 교반하였다. 반응 용액을 여과하고, 테트라하이드로푸란으로 세정하고, 여과액을 감압 농축하였다. 잔류물의 탈수 N,N-디메틸아세트아미드 (25 ㎖) 용액에, 시판 (Aamdis Chemical) 되는 5'-O-[비스(4-메톡시페닐)(페닐)메틸]이노신 (5.0 g) 과 1,8-디아자비시클로[5.4.0]-7-운데센 (2.75 ㎖) 을 첨가하고, 실온에서 3 일간 교반하였다. 반응액에, 물을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (4.41 g) 을 얻었다. To a solution of 3-bromopropan-1-ol (1.14 ml) in tetrahydrofuran (25 ml), triethylamine (1.83 ml) and benzoyl chloride (1.43 ml) were added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was filtered, washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure. To a solution of dehydrated N,N-dimethylacetamide (25 ml) of the residue, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (5.0 g) and 1, 8-Diazabicyclo[5.4.0]-7-undecene (2.75 ml) was added, and the mixture was stirred at room temperature for 3 days. Water was added to the reaction mixture, and extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to obtain the title compound (4.41 g).

Figure pat00339
Figure pat00339

(공정 2) (Process 2)

1-[3-(벤조일옥시)프로필]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]이노신 1-[3-(benzoyloxy)propyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]inosine

상기 공정 1 에서 얻어진 화합물 (4.41 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.60 g) 과 표제 화합물의 위치 이성체인 1-[3-(벤조일옥시)프로필]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]이노신 (1.70 g) 을 얻었다. Using the compound (4.41 g) obtained in step 1 above, reaction was carried out in the same manner as in step 3 of Example 5 to obtain the title compound (1.60 g) and 1-[3-(benzoyloxy), which is a positional isomer of the title compound. This gave propyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]inosine (1.70 g).

Figure pat00340
Figure pat00340

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00341
Figure pat00341

(공정 3) (Process 3)

1-[3-(벤조일옥시)프로필]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}이노신 1-[3-(benzoyloxy)propyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'- O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine

상기 공정 2 에서 얻어진 화합물 (1.60 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.91 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 67 : 33) 로서 얻었다. Using the compound (1.60 g) obtained in step 2 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (1.91 g) was added to a phosphorus atom-phase diastereomer mixture (diastereomer ratio = 67:33). ) was obtained as

Figure pat00342
Figure pat00342

(공정 4) (Process 4)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 981 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 3 에서 얻어진 화합물 (1.03 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 981 mg). Using the acetonitrile solution of the obtained compound and the compound (1.03 g) obtained in the said step 3, reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it was for the next reaction.

(공정 5) (Process 5)

3-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}프로필 벤조에이트 3-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}propyl benzoate

상기 공정 4 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (774 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. A reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 4 above to obtain the title compound (774 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1278(M＋H)＋.MS(ESI) m/z : 1278(M+H) + .

(공정 6) (Process 6)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[1-(3-하이드록시프로필)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- [1-(3-hydroxypropyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetra hydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 5 에서 얻어진 화합물 (774 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, C18 실리카 겔 칼럼 크로마토그래피 [0.2 ％ 트리에틸아민 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (101 ㎎ : 불순물 함유) 과 디아스테레오머 2 (90.8 ㎎ : 불순물 함유) 를 얻었다. Using the compound (774 mg) obtained in step 5 above, reaction was carried out using the same method as in step 10 of Example 1, followed by purification by C18 silica gel column chromatography [0.2% aqueous solution of triethylamine/acetonitrile]. , diastereomer 1 (101 mg: containing impurities) and diastereomer 2 (90.8 mg: containing impurities) of the title compound were obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1017(M＋H)＋. MS(ESI)m/z: 1017(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1017(M＋H)＋.MS(ESI)m/z: 1017(M+H) + .

(공정 7-1) (Process 7-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(3-하이드록시프로필)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(3-hydroxypropyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (101 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 1) (101 mg: containing impurities) obtained in step 6 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (48.8 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (48.8 mg).

Figure pat00343
Figure pat00343

(공정 7-2) (Process 7-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[1-(3-하이드록시프로필)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(3-hydroxypropyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (90.8 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 2) (90.8 mg: containing impurities) obtained in step 6 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 3 ％ - 20 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 3% - 20% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (22.3 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (22.3 mg).

Figure pat00344
Figure pat00344

실시예 37 : CDN27 의 합성 Example 37: Synthesis of CDN27

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-아미노-1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-amino-1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine- 9-yl]-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11 ,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 166][Formula 166]

Figure pat00345
Figure pat00345

[합성 스킴][Synthesis Scheme]

[화학식 167][Formula 167]

Figure pat00346
Figure pat00346

(공정 1) (Process 1)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-N-[(디메틸아미노)메틸리덴]구아노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-N-[(dimethylamino)methylidene]guanosine

이미 알려진 문헌 (Journal of Organic Chemistry, 1994, 59, 7243-7248) 의 N-[(디메틸아미노)메틸리덴]구아노신 (10.0 g) 의 N,N-디메틸아세트아미드 (50 ㎖) - 피리딘 (50 ㎖) 혼합 용액에 4,4'-디메톡시트리틸클로라이드 (10.5 g) 를 0 ℃ 에서 첨가하고, 4 ℃ 에서 16 시간 교반하였다. 반응액에 벤조산2-브로모에틸 (6.54 ㎖) 과 1,8-디아자비시클로[5.4.0]-7-운데센 (11.0 ㎖) 을 첨가하고, 실온에서 2 일간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 물을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물을 트리페닐포스핀옥사이드와의 혼합물 (16.7 g) 로서 얻었다.N, N-dimethylacetamide (50 ml) of N-[(dimethylamino)methylidene]guanosine (10.0 g) from the previously known literature (Journal of Organic Chemistry, 1994, 59, 7243-7248) - pyridine (50 ml) 4,4'-dimethoxytrityl chloride (10.5 g) was added to the mixed solution at 0°C, and the mixture was stirred at 4°C for 16 hours. 2-bromoethyl benzoate (6.54 ml) and 1,8-diazabicyclo[5.4.0]-7-undecene (11.0 ml) were added to the reaction solution, and the mixture was stirred at room temperature for 2 days. A saturated aqueous solution of sodium hydrogen carbonate and water were added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to obtain the title compound as a mixture with triphenylphosphine oxide (16.7 g).

Figure pat00347
Figure pat00347

(공정 2) (Process 2)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-N-[(디메틸아미노)메틸리덴]구아노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-N-[ (dimethylamino)methylidene]guanosine

상기 공정 1 에서 얻어진 화합물 (15.7 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.82 g) 과 표제 화합물의 위치 이성체인 1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-N-[(디메틸아미노)메틸리덴]구아노신 (6.01 g) 을 얻었다.Using the compound (15.7 g) obtained in the above step 1, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (4.82 g) and 1-[2-(benzoyloxy), which is a positional isomer of the title compound. Ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-N-[(dimethylamino)methylidene]guanosine (6.01 g) was obtained.

Figure pat00348
Figure pat00348

위치 이성체 (2'-O-TBS 체) Positional isomers (2'-O-TBS isomers)

Figure pat00349
Figure pat00349

(공정 3) (Process 3)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-N-[(디메틸아미노)메틸리덴]구아노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'- O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-N-[(dimethylamino)methylidene]guanosine

상기 공정 2 에서 얻어진 화합물 (4.81 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.41 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 7 : 3) 로서 얻었다. Using the compound (4.81 g) obtained in step 2 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (5.41 g) was obtained by diastereomer mixture on phosphorus atom (diastereomer ratio = 7:3). ) was obtained as

Figure pat00350
Figure pat00350

(공정 4) (Process 4)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.68 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 3 에서 얻어진 화합물 (1.81 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Example 1 Step 7 was carried out on the following scale (raw material: 1.68 g). Using the acetonitrile solution of the obtained compound and the compound (1.81 g) obtained in the above step 3, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it is for the next reaction.

(공정 5) (Process 5)

2-(9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-2-{(E)-[(디메틸아미노)메틸리덴]아미노}-6-옥소-6,9-디하이드로-1H-푸린-1-일)에틸 벤조에이트 2-(9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7-yl]-2-{(E)-[(dimethylamino)methylidene]amino}-6-oxo-6,9-dihydro-1H-purine- 1-yl)ethyl benzoate

상기 공정 4 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.15 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 4 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (1.15 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1334(M＋H)＋.MS(ESI)m/z: 1334(M+H) + .

(공정 6) (Process 6)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[2-아미노-1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[2-amino-1-(2-hydroxyethyl)-6-oxo -1,6-dihydro-9H-purin-9-yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

상기 공정 5 에서 얻어진 화합물 (1.15 g) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (134 ㎎ : 불순물 함유) 과 디아스테레오머 2 (127 ㎎ : 불순물 함유) 를 얻었다.Using the compound (1.15 g) obtained in step 5 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (134 mg: containing impurities) and diastereomer 2 ( 127 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1018(M＋H)＋. MS(ESI)m/z: 1018(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1018(M＋H)＋.MS(ESI)m/z: 1018(M+H) + .

(공정 7-1) (Process 7-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-아미노-1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-amino-1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H- Purin-9-yl] -15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (134 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (134 mg: containing impurities) obtained in Step 6 above, the reaction was carried out in the same manner as in Step 11 of Example 1, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (36.0 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (36.0 mg).

Figure pat00351
Figure pat00351

(공정 7-2) (Process 7-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-아미노-1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[2-amino-1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H- Purin-9-yl] -15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (127 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (127 mg: containing impurities) obtained in Step 6 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 3 ％ - 20 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 3% - 20% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (20.1 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (20.1 mg).

Figure pat00352
Figure pat00352

실시예 38 : CDN28 의 합성 Example 38: Synthesis of CDN28

N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-2-하이드록시아세트아미드N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10 -bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H ,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl ]-9H-purin-2-yl}amino)ethyl]-2-hydroxyacetamide

[화학식 168][Formula 168]

Figure pat00353
Figure pat00353

[합성 스킴][Synthesis Scheme]

[화학식 169][Formula 169]

Figure pat00354
Figure pat00354

(공정 1) (Process 1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[2-(2-하이드록시아세트아미드)에틸]아미노}-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[2-(2-hydroxyacetamide)ethyl]amino}-9H-purine- 9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 8 공정 8-2 에서 얻어진 화합물 (6.6 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (6.6 mg) obtained in Example 8 Step 8-2, reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (2.9 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (2.9 mg).

Figure pat00355
Figure pat00355

실시예 39 : CDN29 의 합성 Example 39: Synthesis of CDN29

N-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}메틸)-2-하이드록시아세트아미드N-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis( Sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H- 5,8-methano- 2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H -purin-2-yl}methyl)-2-hydroxyacetamide

[화학식 170][Formula 170]

Figure pat00356
Figure pat00356

[합성 스킴][Synthesis Scheme]

[화학식 171][Formula 171]

Figure pat00357
Figure pat00357

(공정 1) (Process 1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-하이드록시아세트아미드)메틸]-9H-푸린-9-일}-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyacetamide)methyl]-9H-purin-9-yl}- 15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

실시예 11 공정 9-2 에서 얻어진 화합물 (4.6 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다.Example 11 Using the compound (4.6 mg) obtained in Step 9-2, the reaction was carried out in the same manner as in Example 7 Step 1-1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile : 2% - 30% (0 min - 30 min)] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (4.0 ㎎) 을 얻었다.The obtained triethylamine salt was subjected to salt exchange in the same manner as described in [Conversion to sodium salt] in Step 11 of Example 1 to obtain the title compound (4.0 mg).

Figure pat00358
Figure pat00358

실시예 40 : CDN30 의 합성 Example 40: Synthesis of CDN30

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl]amino}-9H-purin-9-yl)- 15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 172][Formula 172]

Figure pat00359
Figure pat00359

[합성 스킴][Synthesis Scheme]

[화학식 173][Formula 173]

Figure pat00360
Figure pat00360

(공정 1-1) (Process 1-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-클로로-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-chloro-9H-purin-9-yl)- 15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 8 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (590 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (420 ㎎) 을 얻었다. Example 8 Using the compound (diastereomer 1) obtained in Step 6 (590 mg), a reaction was carried out in the same manner as in Example 1 Step 10 to obtain the title compound (420 mg).

Figure pat00361
Figure pat00361

(공정 1-2) (Process 1-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-클로로-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-chloro-9H-purin-9-yl)- 15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 8 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (710 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (452 ㎎) 을 얻었다. Example 8 Using the compound (diastereomer 2) (710 mg) obtained in Step 6, a reaction was carried out in the same manner as in Example 1 Step 10 to obtain the title compound (452 mg).

Figure pat00362
Figure pat00362

(공정 2-1) (Step 2-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl] Amino}-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro -2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

1-(아미노메틸)시클로프로판-1-아민·2HCl (309 ㎎) 의 메탄올 (40 ㎖) 용액에, MP-Carbonate 레진 (5.45 g) 을 첨가하고, 실온에서 2 시간 교반하였다. 레진을 여과 제거하고, 여과액을 감압 농축하였다. 잔류물의 메탄올 (0.837 ㎖) 용액을, 상기 공정 1-1 에서 얻어진 화합물 (30.0 ㎎) 에 첨가하고, 마이크로 웨이브 반응 장치를 사용하여, 120 ℃ 에서 4 시간 반응시켰다. 반응액을 감압 농축 후, 잔류물을 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 포함하는 혼합물을 얻었다. 얻어진 혼합물은 그대로 다음의 공정에 사용하였다. To a solution of 1-(aminomethyl)cyclopropan-1-amine 2HCl (309 mg) in methanol (40 ml) was added MP-Carbonate resin (5.45 g), and the mixture was stirred at room temperature for 2 hours. The resin was filtered off, and the filtrate was concentrated under reduced pressure. A solution of the residue in methanol (0.837 ml) was added to the compound (30.0 mg) obtained in step 1-1, and the mixture was reacted at 120°C for 4 hours using a microwave reactor. After concentration of the reaction solution under reduced pressure, the residue was purified by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 60% (0 min - 30 min)], and a mixture containing the title compound got The obtained mixture was used as it was in the next step.

MS(ESI)m/z : 1042(M＋H)＋.MS(ESI) m/z : 1042(M+H) + .

(공정 2-2) (Process 2-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl] Amino}-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro -2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 1-2 에서 얻어진 화합물 (58.6 ㎎) 을 사용하여, 상기 공정 2-1 과 동일한 수법에 의해 표제 화합물을 포함하는 혼합물을 얻었다. 얻어진 혼합물은 그대로 다음의 반응에 사용하였다. A mixture containing the title compound was obtained by the same method as in Step 2-1, using the compound (58.6 mg) obtained in Step 1-2 above. The obtained mixture was used as it was for the next reaction.

MS(ESI)m/z : 1042(M＋H)＋.MS(ESI) m/z : 1042(M+H) + .

(공정 3-1) (Step 3-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl]amino}-9H-purin-9-yl )-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene- 2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 2-1 에서 얻어진 혼합물을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the mixture obtained in step 2-1 above, reaction was carried out in the same manner as in step 11 of Example 1, and then preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (2.0 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (2.0 mg).

Figure pat00363
Figure pat00363

(공정 3-2) (Step 3-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl] amino} -9H-purin-9-yl) -15,16-dihydroxy-2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6 -Tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6 ,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 2-2 에서 얻어진 혼합물을 사용하여, 실시예 1 공정 11 과 동일한 수법에 의해 표제 화합물을 포함하는 혼합물을 얻었다. 얻어진 혼합물은 그대로 다음의 반응에 사용하였다.A mixture containing the title compound was obtained by the same method as in Example 1 Step 11 using the mixture obtained in the above step 2-2. The obtained mixture was used as it was for the next reaction.

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-아미노-2-({[1-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)시클로프로필]메틸}아미노)-9H-푸린-9-일]-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-({[1-({[2-( trimethylsilyl)ethoxy]carbonyl}amino)cyclopropyl]methyl}amino)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7, 8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 3-2 에서 얻어진 혼합물의 N,N-디메틸포름아미드 (1.0 ㎖) 용액에 트리에틸아민 (40.8 ㎕) 과 2,5-디옥소피롤리딘-1-일(2-(트리메틸실릴)에틸)카보네이트 (39.5 ㎎) 를 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 물을 첨가하여 퀀치 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 50 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (11.0 ㎎) 을 얻었다. Triethylamine (40.8 μl) and 2,5-dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl )carbonate (39.5 mg) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched by adding water, and then purified by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 50% (0 min - 30 min)] to obtain the title compound (11.0 mg) got

Figure pat00364
Figure pat00364

(공정 5) (Process 5)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[(1-아미노시클로프로필)메틸]아미노}-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[(1-aminocyclopropyl)methyl]amino}-9H-purin-9-yl )-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene- 2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 4 에서 얻어진 화합물 (11.0 ㎎) 의 테트라하이드로푸란 (474 ㎕) 용액에, 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 237 ㎕) 을 첨가하고, 질소 분위기하, 40 ℃ 에서 3 시간 교반하였다. 반응액에 10 mM 아세트산트리에틸암모늄 수용액을 첨가하여 퀀치 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)] 및 Sep-Pak (등록상표) C18 [물/아세토니트릴/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. To a solution of the compound (11.0 mg) obtained in step 4 above in tetrahydrofuran (474 μl), a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 237 μl) was added, and the mixture was stirred at 40° C. in a nitrogen atmosphere for 3 Stir for an hour. After quenching by adding 10 mM triethylammonium acetate aqueous solution to the reaction solution, preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)] and Sep-Pak (registered trademark) C18 [water/acetonitrile/0.1% triethylamine] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (4.1 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (4.1 mg).

Figure pat00365
Figure pat00365

실시예 41 : CDN31 의 합성 Example 41: Synthesis of CDN31

(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[2-(하이드록시메틸)-6-(메틸아미노)-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[2-(hydroxymethyl)-6-(methylamino)-9H-purine-9- yl] -2,10-bis (sulfanyl) -14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecyne-2,10-dione

[화학식 174][Formula 174]

Figure pat00366
Figure pat00366

[합성 스킴][Synthesis Scheme]

[화학식 175][Formula 175]

Figure pat00367
Figure pat00367

(공정 1) (Process 1)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-[2-(하이드록시메틸)-6-(메틸아미노)-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[2-(hydroxymethyl)-6-( Methylamino)-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11 ,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

실시예 12 공정 7 에서 얻어진 디아스테레오머 2 (고극성) (56.5 ㎎) 의 메탄올 (1.00 ㎖) 용액에, 40 ％ 메틸아민 수용액 (1.00 ㎖) 을 첨가하고, 봉관 (封管) 중 60 ℃ 에서 3 시간 교반하였다. 반응액을 감압 농축하여 표제 화합물을 포함하는 혼합물을 얻었다. 얻어진 혼합물을 그대로 다음의 반응에 사용하였다.Example 12 To a solution of diastereomer 2 (high polarity) (56.5 mg) obtained in Step 7 in methanol (1.00 mL) was added 40% methylamine aqueous solution (1.00 mL), and the mixture was stirred at 60°C in a sealed tube. Stir for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a mixture containing the title compound. The obtained mixture was used as it was for the next reaction.

MS(ESI)m/z : 1002(M＋H)＋.MS(ESI) m/z : 1002(M+H) + .

(공정 2) (Process 2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-7-[2-(하이드록시메틸)-6-(메틸아미노)-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[2-(hydroxymethyl)-6-(methylamino)-9H-purine- 9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyne-2,10-bis(thiolate)

상기 공정 1 에서 얻어진 혼합물을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 20 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. After reacting by the same method as in Example 1 step 11 using the mixture obtained in step 1 above, preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 20% (0 min - 30 min)] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (16.0 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (16.0 mg).

Figure pat00368
Figure pat00368

실시예 42 : CDN32 의 합성 Example 42: Synthesis of CDN32

(5R,7R,8R,12aR,14R,15aS,16R)-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15aS,16R)-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine-9 -yl] -2,10-bis (sulfanyl) -14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

[화학식 176][Formula 176]

Figure pat00369
Figure pat00369

[합성 스킴][Synthesis Scheme]

[화학식 177][Formula 177]

Figure pat00370
Figure pat00370

(공정 1) (Process 1)

5-(3,3-디에톡시프로파-1-인-1-일)-7H-피롤로[2,3-d]피리미딘-4-아민 5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

시판 (PharmaBlock) 되는 5-요오드-7H-피롤로[2,3-d]피리미딘-4-아민 (22 g) 의 N,N-디메틸포르미아미드 (70 ㎖) 용액에, 요오드화구리 (1.61 g), 비스(트리페닐포스핀)팔라듐디클로라이드 (5.94 g), 및 트리에틸아민 (35 ㎖) 을 첨가하였다. 프로파르길알데히드디에틸아세탈 (22 ㎖) 을 2 시간 걸쳐 첨가하고, 실온에서 밤새 교반하였다. 반응액에 클로로포름 (350 ㎖) 을 첨가하고, 물로 2 회 세정하였다. 유기층을 황산마그네슘으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 아세트산에틸 (350 ㎖) 을 첨가하고, 밤새 교반하였다. 석출한 고체를 여과 채취하고, 표제 화합물 (9.60 g) 을 얻었다. To a solution of commercially available (PharmaBlock) 5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine (22 g) in N,N-dimethylformiamide (70 ml), copper iodide (1.61 g), bis(triphenylphosphine)palladiumdichloride (5.94 g), and triethylamine (35 mL) were added. Propargylaldehydediethylacetal (22 mL) was added over 2 hours and stirred at room temperature overnight. Chloroform (350 ml) was added to the reaction mixture, and the mixture was washed twice with water. After drying the organic layer with magnesium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethyl acetate (350 mL) was added to the residue and stirred overnight. The precipitated solid was collected by filtration to obtain the title compound (9.60 g).

Figure pat00371
Figure pat00371

(공정 2) (Process 2)

5-(3,3-디에톡시프로필)-7H-피롤로[2,3-d]피리미딘-4-아민 5-(3,3-diethoxypropyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 1 에서 얻어진 화합물 (17.9 g) 의 테트라하이드로푸란 (160 ㎖) - 에탄올 (80 ㎖) 혼합 용액에, 10 ％ 팔라듐탄소 (M) wet (25.0 g) 를 첨가하고, 수소 분위기하, 실온에서 밤새 교반하였다. 촉매를 셀라이트로 여과 제거하고, 여과액을 감압 농축하고, 표제 화합물 (17.0 g) 의 미정제체를 얻었다. 10% palladium carbon (M) wet (25.0 g) was added to a tetrahydrofuran (160 ml)-ethanol (80 ml) mixed solution of the compound (17.9 g) obtained in step 1 above, and the mixture was heated at room temperature under a hydrogen atmosphere. Stir overnight. The catalyst was filtered off through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound (17.0 g).

MS(ESI)m/z : 265(M＋H)＋.MS(ESI) m/z : 265(M+H) + .

(공정 3) (Process 3)

6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 2 에서 얻어진 화합물 (50.21 g) 을 90 ％ 아세트산 수용액 (344 ㎖) 에 용해하고, 50 ℃ 에서 밤새 교반하였다. 원료의 소실을 확인 후, 반응액에 팔라듐탄소 (M) wet (60 g) 를 첨가하고, 수소 분위기하, 40 ℃ 에서 밤새 교반하였다. 촉매를 셀라이트로 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 포화 탄산수소나트륨 수용액 (350 ㎖) 을 첨가하고, 클로로포름/메탄올 (9 : 1) 로 7 회 추출하였다. 유기층을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하고, 표제 화합물 (18.47 g) 을 얻었다. The compound (50.21 g) obtained in the above step 2 was dissolved in a 90% aqueous acetic acid solution (344 ml), and the mixture was stirred at 50°C overnight. After confirming the disappearance of the raw materials, palladium carbon (M) wet (60 g) was added to the reaction mixture, and the mixture was stirred overnight at 40°C under a hydrogen atmosphere. The catalyst was filtered off through celite, and the filtrate was concentrated under reduced pressure. Saturated sodium hydrogencarbonate aqueous solution (350 ml) was added to the residue, and extraction was performed 7 times with chloroform/methanol (9:1). After concentration of the organic layer under reduced pressure, the residue was purified by silica gel column chromatography [chloroform/methanol] to obtain the title compound (18.47 g).

Figure pat00372
Figure pat00372

(공정 4) (Process 4)

페닐(2,7,8,9-테트라하이드로-6H-2,3,5,6-테트라아자벤조[cd]아줄렌-6-일)메타논 Phenyl(2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulen-6-yl)methanone

상기 공정 3 에서 얻어진 화합물 (8.47 g) 의 디클로로메탄 (120 ㎖) 현탁액에, 탈수 피리딘 (39.2 ㎖), N,N-디메틸아미노피리딘 (2.38 g), 및 염화벤조일 (22.6 ㎖) 을 차례로 첨가하고, 실온에서 1 시간 교반하였다. 반응액을 감압 농축 후, 잔류물에 클로로포름 (150 ㎖), 메탄올 (60 ㎖), 및 트리에틸아민 (50 ㎖) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액을 클로로포름과 물의 2 층에 따르고, 클로로포름으로 추출하였다. 유기층을 25 w/v％ 황산수소칼륨 수용액으로 2 회 세정하고, 무수 황산마그네슘으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 아세트산에틸 (50 ㎖) 과 헥산 (125 ㎖) 을 차례로 첨가하여 슬러리상으로 한 후, 1 시간 교반하였다. 석출한 고체를 여과 채취하고, 표제 화합물 (9.89 g) 을 얻었다. To a suspension of the compound (8.47 g) obtained in step 3 in dichloromethane (120 ml), dehydrated pyridine (39.2 ml), N,N-dimethylaminopyridine (2.38 g), and benzoyl chloride (22.6 ml) were added in this order, , and stirred at room temperature for 1 hour. After concentrating the reaction solution under reduced pressure, chloroform (150 ml), methanol (60 ml) and triethylamine (50 ml) were added to the residue, and the mixture was stirred at room temperature for 3 hours. The reaction solution was poured into two layers of chloroform and water, and extracted with chloroform. The organic layer was washed twice with a 25 w/v% aqueous solution of potassium hydrogen sulfate and dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate (50 ml) and hexane (125 ml) were successively added to the residue to form a slurry, followed by stirring for 1 hour. The precipitated solid was collected by filtration to obtain the title compound (9.89 g).

Figure pat00373
Figure pat00373

(공정 5) (Process 5)

6-벤조일-2-[2-데옥시-3,5-비스-O-(4-메틸벤조일)-β-D-에리트로-펜토퓨라노실]-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-[2-deoxy-3,5-bis-O-(4-methylbenzoyl)-β-D-erythro-pentofuranosyl]-6,7,8,9-tetrahydro- 2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (7.10 g) 의 아세토니트릴 (80 ㎖) 현탁액에, 질소 분위기하, 분말상의 수산화칼륨 (2.9 g) 과 트리스[2-(2-메톡시에톡시)에틸]아민 (0.41 ㎖) 을 첨가하고, 실온에서 15 분간 교반하였다. 빙랭하, 이미 알려진 문헌 (Synlett 2004 (2) : 335-337) 의 2-데옥시-3,5-비스-O-(4-메틸벤조일)-α-D-에리트로-펜토퓨라노실클로라이드 (10.12 g) 와 아세토니트릴 (60 ㎖) 을 첨가하고, 실온까지 승온하고, 밤새 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하여 반응을 정지시켰다. 석출한 고체를 여과 채취한 후, 물로 세정하고, 표제 화합물 (9.70 g) 을 얻었다. To a suspension of the compound (7.10 g) obtained in step 4 in acetonitrile (80 ml), powdery potassium hydroxide (2.9 g) and tris[2-(2-methoxyethoxy)ethyl]amine (0.41 ml) was added and stirred at room temperature for 15 minutes. Under ice cooling, 2-deoxy-3,5-bis-O-(4-methylbenzoyl)-α-D-erythro-pentofuranosyl chloride (Synlett 2004 (2): 335-337) 10.12 g) and acetonitrile (60 ml) were added, the temperature was raised to room temperature, and the mixture was stirred overnight. A saturated aqueous solution of ammonium chloride was added to the reaction solution to stop the reaction. The precipitated solid was collected by filtration and washed with water to obtain the title compound (9.70 g).

Figure pat00374
Figure pat00374

(공정 6) (Process 6)

6-벤조일-2-(2-데옥시-β-D-에리트로-펜토퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-(2-deoxy-β-D-erythro-pentofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd ]azulene

상기 공정 5 에서 얻어진 화합물 (8.69 g) 의 메탄올 (45 ㎖) - 테트라하이드로푸란 (135 ㎖) 혼합 용액에, -10 ℃ 에서 2 규정 수산화나트륨 수용액 (27.6 ㎖) 을 20 분간 걸쳐 적하하였다. 동 온도에서 2 시간 교반 후, 1 규정 염산 (58 ㎖) 을 첨가하여 반응을 정지시켰다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하고, 표제 화합물 (4.09 g) 을 얻었다.To a mixed solution of the compound (8.69 g) obtained in step 5 above in methanol (45 ml) - tetrahydrofuran (135 ml), 2N aqueous sodium hydroxide solution (27.6 ml) was added dropwise over 20 minutes at -10°C. After stirring at the same temperature for 2 hours, 1 normal hydrochloric acid (58 ml) was added to stop the reaction. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [chloroform/methanol] to obtain the title compound (4.09 g).

Figure pat00375
Figure pat00375

(공정 7) (Process 7)

6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-데옥시-β-D-에리트로-펜토퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-erythro-pentofuranosyl}-6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 6 에서 얻어진 화합물 (4.55 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (6.38 g) 을 얻었다. Using the compound (4.55 g) obtained in the above step 6, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (6.38 g).

Figure pat00376
Figure pat00376

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 8) (Process 8)

6-벤조일-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-데옥시-β-D-에리트로-펜토퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino] Phosphanyl} -2-deoxy-β-D-erythro-pentofuranosyl) -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 7 에서 얻어진 화합물 (6.37 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (6.05 g) 을 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (6.37 g) obtained in step 7 above, reaction was carried out in the same manner as in step 6 of Example 1 to obtain the title compound (6.05 g) as a diastereomer mixture (diastereomer ratio = 1:1). .

Figure pat00377
Figure pat00377

(공정 9) (Process 9)

상기 공정 8 에서 얻어진 화합물 (868 ㎎) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-에리트로-펜토퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 실시예 22 공정 3 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (868 mg) obtained in step 8 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 6-benzoyl-2-{2-deoxy-3-O-[hydroxy(oxo) -λ 5 -phosphanyl] -β-D-erythro-pentofuranosyl} -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene An acetonitrile solution was obtained. Using this acetonitrile solution and the compound (1.00 g) obtained in Example 22 Step 3, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 10) (Process 10)

2-{9-[(5R,7R,8R,12aR,14R,15aS,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15aS,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10 -sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 9 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (702 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 9 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (702 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1134(M＋H)＋.MS(ESI)m/z: 1134(M+H) + .

(공정 11) (Process 11)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-Diethylethanenaminium) (5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2 -Hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2 ,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 10 에서 얻어진 화합물 (702 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (119 ㎎ : 불순물 함유) 과 디아스테레오머 2 (113 ㎎ : 불순물 함유) 를 얻었다.Using the compound (702 mg) obtained in step 10 above, reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (119 mg: impurity containing) and diastereomer 2 ( 113 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 873(M＋H)＋. MS(ESI) m/z : 873(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 873(M＋H)＋.MS(ESI) m/z : 873(M+H) + .

(공정 12-1) (Process 12-1)

디나트륨 (5R,7R,8R,12aR,14R,15aS,16R)-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15aS,16R)-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine -9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 1) (119 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (119 mg: containing impurities) obtained in Step 11 above, reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (51.8 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (51.8 mg).

Figure pat00378
Figure pat00378

(공정 12-2) (Process 12-2)

디나트륨 (5R,7R,8R,12aR,14R,15aS,16R)-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15aS,16R)-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine -9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 2) (113 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (113 mg: containing impurities) obtained in Step 11 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (65.4 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (65.4 mg).

Figure pat00379
Figure pat00379

실시예 43 : CDN33 의 합성 Example 43: Synthesis of CDN33

(5R,7R,8R,12aR,14R,15S,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15S,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 178][Formula 178]

Figure pat00380
Figure pat00380

[합성 스킴][Synthesis Scheme]

[화학식 179][Formula 179]

Figure pat00381
Figure pat00381

(공정 1) (Process 1)

1-(2,7,8,9-테트라하이드로-6H-2,3,5,6-테트라아자벤조[cd]아줄렌-6-일)에탄-1-온 1-(2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulen-6-yl)ethan-1-one

실시예 42 공정 3 에서 얻어진 화합물 (6.88 g) 을 무수 아세트산 (48 ㎖) 에 용해하고, 90 ℃ 에서 교반하였다. 반응액을 감압 농축하고, 잔류물을 클로로포름 (100 ㎖) - 메탄올 (50 ㎖) - 트리에틸아민 (30 ㎖) 의 혼합액에 용해하였다. 실온에서 4 시간 교반 후, 반응액을 클로로포름과 물의 2 층에 따르고, 클로로포름으로 추출하였다. 유기층을 황산마그네슘으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 헥산/아세트산에틸 (1 : 2) 을 첨가하여 슬러리상으로 한 후, 고체를 여과 채취하고, 표제 화합물 (7.18 g) 을 얻었다. The compound (6.88 g) obtained in Example 42 step 3 was dissolved in acetic anhydride (48 ml) and stirred at 90°C. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in a mixture of chloroform (100 ml) - methanol (50 ml) - triethylamine (30 ml). After stirring at room temperature for 4 hours, the reaction solution was poured into two layers of chloroform and water, followed by extraction with chloroform. After drying the organic layer with magnesium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Hexane/ethyl acetate (1:2) was added to the residue to form a slurry, and then the solid was collected by filtration to obtain the title compound (7.18 g).

Figure pat00382
Figure pat00382

(공정 2) (Process 2)

6-아세틸-2-(3,5-디-O-벤조일-2-데옥시-2-플루오로-β-D-아라비노퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌6-Acetyl-2-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl)-6,7,8,9-tetrahydro-2H -2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 1 에서 얻어진 화합물 (6.00 g) 과 시판 (CARBOSYNTH) 되는 [(2R,3R,4S,5R)-3-벤조일옥시-5-브로모-4-플루오로-테트라하이드로푸란-2-일]메틸벤조에이트 (14.1 g) 를 사용하여, 실시예 42 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (11.45 g) 을 얻었다. The compound obtained in step 1 (6.00 g) and commercially available (CARBOSYNTH) [(2R,3R,4S,5R)-3-benzoyloxy-5-bromo-4-fluoro-tetrahydrofuran-2-yl] The reaction was carried out in the same manner as in Example 42 step 5 using methyl benzoate (14.1 g) to obtain the title compound (11.45 g).

Figure pat00383
Figure pat00383

(공정 3) (Process 3)

6-아세틸-2-(2-데옥시-2-플루오로-β-D-아라비노퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-Acetyl-2-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetra azabenzo[cd]azulene

상기 공정 2 에서 얻어진 화합물 (8.70 g) 의 메탄올 (58 ㎖) - 테트라하이드로푸란 (117 ㎖) 혼합 용액에, -20 ℃ 에서 2 규정 수산화나트륨 수용액 (32 ㎖) 을 12 분간 걸쳐 적하하였다. 동 온도에서 3 시간 교반 후, 1 규정 염산 (66 ㎖) 을 첨가하여 반응을 정지시켰다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하고, 표제 화합물 (3.27 g) 을 얻었다. To a mixed solution of the compound (8.70 g) obtained in step 2 above in methanol (58 ml) and tetrahydrofuran (117 ml), 2N aqueous sodium hydroxide solution (32 ml) was added dropwise over 12 minutes at -20°C. After stirring at the same temperature for 3 hours, 1 normal hydrochloric acid (66 ml) was added to stop the reaction. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [chloroform/methanol] to obtain the title compound (3.27 g).

Figure pat00384
Figure pat00384

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 4) (Process 4)

6-아세틸-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-데옥시-2-플루오로-β-D-아라비노퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-Acetyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-6,7 ,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 3 에서 얻어진 화합물 (3.95 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.66 g) 을 얻었다.Using the compound (3.95 g) obtained in the above step 3, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (5.66 g).

Figure pat00385
Figure pat00385

(공정 5) (Process 5)

6-아세틸-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-데옥시-2-플루오로-β-D-아라비노퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-acetyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino] Phosphanyl} -2-deoxy-2-fluoro-β-D-arabinofuranosyl) -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ cd]azulene

상기 공정 4 에서 얻어진 화합물 (5.68 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.08 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다.Using the compound (5.68 g) obtained in step 4 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (5.08 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00386
Figure pat00386

(공정 6) (Process 6)

상기 공정 5 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-아세틸-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-아라비노퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 이 아세토니트릴 용액과 실시예 22 공정 3 에서 얻어진 화합물 (1.21 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.00 g) obtained in step 5 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 6-acetyl-2-{2-deoxy-2-fluoro-3-O-[ Hydroxy (oxo)-λ 5 -phosphanyl] -β-D-arabinofuranosyl} -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd ]Obtained a solution of azulene in acetonitrile. Using this acetonitrile solution and the compound (1.21 g) obtained in Example 22 Step 3, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 7) (Process 7)

2-{9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-아세틸-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-acetyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-oxo -2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 6 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (757 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 6 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (757 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1090(M＋H)＋.MS(ESI) m/z : 1090(M+H) + .

(공정 8) (Process 8)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15S,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15S,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 7 에서 얻어진 화합물 (757 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (113 ㎎ : 불순물 함유) 과 디아스테레오머 2 (108 ㎎ : 불순물 함유) 를 얻었다.Using the compound (757 mg) obtained in step 7 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (113 mg: impurity containing) and diastereomer 2 ( 108 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 891(M＋H)＋. MS(ESI) m/z : 891(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 891(M＋H)＋.MS(ESI) m/z : 891(M+H) + .

(공정 9-1) (Process 9-1)

디나트륨 (5R,7R,8R,12aR,14R,15S,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15S,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 1) (113 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (113 mg: containing impurities) obtained in Step 8 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (28.8 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (28.8 mg).

Figure pat00387
Figure pat00387

(공정 9-2) (Process 9-2)

디나트륨 (5R,7R,8R,12aR,14R,15S,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15S,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 2) (108 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (108 mg: containing impurities) obtained in Step 8 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 20 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 20% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (3.2 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (3.2 mg).

Figure pat00388
Figure pat00388

실시예 44 : CDN34 의 합성 Example 44: Synthesis of CDN34

(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 180][Formula 180]

Figure pat00389
Figure pat00389

[합성 스킴][Synthesis Scheme]

[화학식 181][Formula 181]

Figure pat00390
Figure pat00390

(공정 1) (Process 1)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulene

실시예 1 공정 4 에서 얻어진 화합물 (35.80 g) 의 디클로로메탄 (322 ㎖) - 피리딘 (35 ㎖) 혼합 용액에, 빙랭하에서 불화수소-피리딘 (6.33 g) 의 디클로로메탄 (36 ㎖) 용액을 5 분간 걸쳐 첨가하고, 동 온도에서 3 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액 (268 ㎖) 과 포화 식염수 (143 ㎖) 를 차례로 첨가하여 반응을 정지시키고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 헥산/아세트산에틸 (1 : 1) (108 ㎖) 을 첨가하여 슬러리상으로 한 후, 50 ℃ 에서 30 분간 교반하고, 헥산 (161 ㎖) 을 추가하여 2 시간 더 교반하였다. 석출한 고체를 여과 채취하고, 헥산/아세트산에틸 (4 : 1) (143 ㎖) 로 세정하고, 표제 화합물 (26.81 g) 을 얻었다. Example 1 To a dichloromethane (322 mL)-pyridine (35 mL) mixed solution of the compound (35.80 g) obtained in Step 4, a solution of hydrogen fluoride-pyridine (6.33 g) in dichloromethane (36 mL) was added to the solution under ice-cooling for 5 minutes. It was added over and stirred at the same temperature for 3 hours. Saturated sodium hydrogen carbonate aqueous solution (268 ml) and saturated brine (143 ml) were sequentially added to the reaction mixture to stop the reaction, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Hexane/ethyl acetate (1:1) (108 mL) was added to the residue to form a slurry, and the mixture was stirred at 50°C for 30 minutes, then hexane (161 mL) was added and the mixture was further stirred for 2 hours. The precipitated solid was collected by filtration and washed with hexane/ethyl acetate (4:1) (143 ml) to obtain the title compound (26.81 g).

Figure pat00391
Figure pat00391

(공정 2) (Process 2)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3,5-비스-O-(옥산-2-일)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl}-6,7 ,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 1 에서 얻어진 화합물 (19.93 g) 과 3,4-디하이드로-2H-피란 (35 ㎖) 의 N,N-디메틸포르미아미드 (200 ㎖) 용액에, 빙랭하에서 p-톨루엔술폰산 1수화물 (7.25 g) 을 첨가하고, 실온에서 3 시간 교반하였다. 빙랭하에서 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시키고, 아세트산에틸로 추출하였다. 유기층을 물과 포화 식염수로 차례로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (24.73 g) 을 얻었다. To a solution of the compound (19.93 g) obtained in step 1 above and 3,4-dihydro-2H-pyran (35 ml) in N,N-dimethylformiamide (200 ml) under ice-cooling, p-toluenesulfonic acid monohydrate ( 7.25 g) was added and stirred at room temperature for 3 hours. The reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction mixture under ice-cooling, followed by extraction with ethyl acetate. The organic layer was washed successively with water and brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (24.73 g).

Figure pat00392
Figure pat00392

(공정 3) (Process 3)

6-벤조일-2-[3,5-비스-O-(옥산-2-일)-β-D-리보퓨라노실]-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-[3,5-bis-O-(oxane-2-yl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3; 5,6-tetraazabenzo[cd]azulene

상기 공정 2 에서 얻어진 화합물 (24.73 g) 과 아세트산 (3.1 ㎖) 의 테트라하이드로푸란 (250 ㎖) 용액에, 빙랭하에서 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 55 ㎖) 을 첨가하고, 실온에서 밤새 교반하였다. 반응액을 감압 농축하고, 잔류물에 아세트산에틸을 첨가하고, 물과 포화 식염수로 차례로 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (18.74 g) 을 얻었다. To a solution of the compound (24.73 g) and acetic acid (3.1 ml) obtained in step 2 above in tetrahydrofuran (250 ml), a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 55 ml) was added under ice-cooling, Stir overnight at room temperature. The reaction solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed sequentially with water and saturated brine. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (18.74 g).

Figure pat00393
Figure pat00393

(공정 4) (Process 4)

6-벤조일-2-[3,5-비스-O-(옥산-2-일)-β-D-아라비노퓨라노실]-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-[3,5-bis-O-(oxane-2-yl)-β-D-arabinofuranosyl]-6,7,8,9-tetrahydro-2H-2,3 ,5,6-tetraazabenzo[cd]azulene

상기 공정 3 에서 얻어진 화합물 (18.74 g) 과 피리딘 (13.1 ㎖) 의 디클로로메탄 (300 ㎖) 용액에, 빙랭하에서 트리플루오로메탄술폰산 무수물 (11 ㎖) 을 적하하고 10 분간 교반하였다. 반응액에 포화 식염수를 첨가하여 반응을 정지시키고, 디클로로메탄으로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 테트라하이드로푸란 (300 ㎖) 에 용해하고, 빙랭하에서 아질산테트라부틸암모늄 (28.34 g) 의 테트라하이드로푸란 (150 ㎖) 용액을 적하하고, 실온에서 밤새 교반하였다. 반응액을 감압 농축하고, 잔류물에 아세트산에틸을 첨가하고, 물과 포화 식염수로 차례로 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (10.46 g) 을 얻었다. Trifluoromethanesulfonic anhydride (11 ml) was added dropwise to a dichloromethane (300 ml) solution of the compound (18.74 g) obtained in step 3 and pyridine (13.1 ml) under ice-cooling, followed by stirring for 10 minutes. Saturated brine was added to the reaction solution to stop the reaction, extraction was performed with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (300 ml), and a solution of tetrabutylammonium nitrite (28.34 g) in tetrahydrofuran (150 ml) was added dropwise under ice-cooling, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed sequentially with water and saturated brine. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (10.46 g).

Figure pat00394
Figure pat00394

(공정 5) (Process 5)

6-벤조일-2-[2-데옥시-2-플루오로-3,5-비스-O-(옥산-2-일)-β-D-리보퓨라노실]-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-[2-deoxy-2-fluoro-3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl]-6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (10.46 g) 과 피리딘 (7.3 ㎖) 의 디클로로메탄 (200 ㎖) 용액에, 빙랭하에서 트리플루오로메탄술폰산 무수물 (6.1 ㎖) 을 적하하고 10 분간 교반하였다. 반응액에 포화 식염수를 첨가하여 반응을 정지시키고, 디클로로메탄으로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 테트라하이드로푸란 (200 ㎖) 에 용해하고, 빙랭하에서 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 150 ㎖) 을 첨가하고, 동 온도에서 3 시간 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (7.65 g) 을 얻었다. Trifluoromethanesulfonic anhydride (6.1 ml) was added dropwise to a dichloromethane (200 ml) solution of the compound (10.46 g) obtained in step 4 and pyridine (7.3 ml) under ice-cooling, followed by stirring for 10 minutes. Saturated brine was added to the reaction solution to stop the reaction, extraction was performed with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (200 ml), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 150 ml) was added under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. A saturated aqueous solution of ammonium chloride was added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (7.65 g).

Figure pat00395
Figure pat00395

(공정 6) (Process 6)

6-벤조일-2-(2-데옥시-2-플루오로-β-D-리보퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza benzo[cd]azulene

상기 공정 5 에서 얻어진 화합물 (7.65 g) 의 에탄올 (150 ㎖) 용액에, p-톨루엔술폰산피리디늄 (6.62 g) 을 첨가하고, 50 ℃ 에서 3 시간 교반하였다. 반응액을 감압 농축하고, 잔류물에 아세트산에틸을 첨가하고, 포화 탄산수소나트륨 수용액과 포화 식염수로 차례로 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (3.55 g) 을 얻었다. Pyridinium p-toluenesulfonate (6.62 g) was added to a solution of the compound (7.65 g) obtained in step 5 in ethanol (150 ml), and the mixture was stirred at 50°C for 3 hours. The reaction solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed with saturated sodium hydrogencarbonate aqueous solution and brine in that order. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (3.55 g).

Figure pat00396
Figure pat00396

(공정 7) (Process 7)

6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-데옥시-2-플루오로-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-ribofuranosyl}-6,7, 8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 6 에서 얻어진 화합물 (3.55 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.77 g) 을 얻었다. Using the compound (3.55 g) obtained in the above step 6, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (5.77 g).

Figure pat00397
Figure pat00397

(공정 8) (Process 8)

6-벤조일-2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-데옥시-2-플루오로-β-D-리보퓨라노실)-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino] Phosphanyl} -2-deoxy-2-fluoro-β-D-ribofuranosyl) -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd ]azulene

상기 공정 7 에서 얻어진 화합물 (5.77 g) 을 사용하여, 실시예 1 의 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.95 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (5.77 g) obtained in step 7 above, a reaction was carried out in the same manner as in step 6 of Example 1 to obtain the title compound (5.95 g) on a phosphorus atom-phase diastereomer mixture (diastereomer ratio = 1: 1) was obtained as

Figure pat00398
Figure pat00398

(공정 9) (Process 9)

상기 공정 8 에서 얻어진 화합물 (1.02 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 22 공정 3 에서 얻어진 화합물 (1.15 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.02 g) obtained in step 8 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[ Hydroxy (oxo)-λ 5 -phosphanyl] -β-D-ribofuranosyl} -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] A solution of azulene in acetonitrile was obtained. Using the obtained acetonitrile solution and the compound (1.15 g) obtained in Example 22 step 3, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it was for the next reaction.

(공정 10) (Process 10)

2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-oxo -2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 9 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (818 ㎎ : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 9 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (818 mg: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1152(M＋H)＋.MS(ESI)m/z: 1152(M+H) + .

(공정 11) (Process 11)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 10 에서 얻어진 화합물 (818 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (107 ㎎ : 불순물 함유) 과 디아스테레오머 2 (101 ㎎ : 불순물 함유) 를 얻었다.Using the compound (818 mg) obtained in step 10 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (107 mg: impurity containing) and diastereomer 2 ( 101 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 891(M＋H)＋. MS(ESI) m/z: 891(M+H) +.

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 891(M＋H)＋.MS(ESI) m/z : 891(M+H) + .

(공정 12-1) (Process 12-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 1) (107 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (107 mg: containing impurities) obtained in Step 11 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (29.1 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (29.1 mg).

Figure pat00399
Figure pat00399

(공정 12-2) (Process 12-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 11 에서 얻어진 화합물 (디아스테레오머 2) (101 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (101 mg: containing impurities) obtained in Step 11 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 20 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 20% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (11.2 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (11.2 mg).

Figure pat00400
Figure pat00400

실시예 45 : CDN35 의 합성 Example 45: Synthesis of CDN35

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15-플루오로-16-하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 15-fluoro-16-hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 182][Formula 182]

Figure pat00401
Figure pat00401

[합성 스킴][Synthesis Scheme]

[화학식 183][Formula 183]

Figure pat00402
Figure pat00402

(공정 1) (Process 1)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl ]Inosine

시판 (Aamdis Chemical) 되는 5'-O-[비스(4-메톡시페닐)(페닐)메틸]이노신 (13.0 g) 의 N,N-디메틸아세트아미드 (60 ㎖) 현탁액에, N-(2-브로모에틸)프탈이미드 (7.02 g) 와 1,8-디아자비시클로[5.4.0]-7-운데센 (4.1 ㎖) 을 첨가하고, 실온에서 밤새 교반하였다. N-(2-브로모에틸)프탈이미드 (1.75 g) 와 1,8-디아자비시클로[5.4.0]-7-운데센 (1.1 ㎖) 을 추가하고, 1 일간 더 교반하였다. 반응액에 물을 첨가하여 반응을 정지시키고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (12.4 g) 을 얻었다. To a suspension of commercially available (Aamdis Chemical) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (13.0 g) in N,N-dimethylacetamide (60 ml), N-(2- Bromoethyl)phthalimide (7.02 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (4.1 ml) were added, and the mixture was stirred at room temperature overnight. N-(2-bromoethyl)phthalimide (1.75 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (1.1 ml) were added, and the mixture was stirred for another 1 day. Water was added to the reaction solution to stop the reaction, and extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate/methanol] to obtain the title compound (12.4 g).

Figure pat00403
Figure pat00403

(공정 2) (Process 2)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-[2-(1,3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) ethyl] inosine

상기 공정 1 에서 얻어진 화합물 (12.4 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.18 g) 과 표제 화합물의 위치 이성체인 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]이노신 (6.31 g) 을 얻었다.Using the compound (12.4 g) obtained in the above step 1, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (4.18 g) and 5'-O-[bis(4), which is a positional isomer of the title compound. -methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindole -2-yl)ethyl]inosine (6.31 g) was obtained.

Figure pat00404
Figure pat00404

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00405
Figure pat00405

(공정 3) (Process 3)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine

상기 공정 2 에서 얻어진 화합물 (8.89 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (9.45 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (8.89 g) obtained in step 2 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (9.45 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00406
Figure pat00406

(공정 4) (Process 4)

실시예 44 공정 8 에서 얻어진 화합물 (1.30 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 상기 공정 3 에서 얻어진 화합물 (1.50 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.30 g) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (1.50 g) obtained in the above step 3, a reaction was carried out in the same manner as in step 8 of Example 1, and the obtained crude product was used as it was for the next reaction.

(공정 5) (Process 5)

3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-7-{1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-일]옥시}프로판니트릴 3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-{1-[2-(1,3-dioxo-1,3-di Hydro-2H-isoindol-2-yl)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-15-fluoro-2-oxo-2-sulfanyl-10- Sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa diphosphacyclotetradecin-10-yl]oxy}propanenitrile

상기 공정 4 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (828 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. The reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 4 above to obtain the title compound (828 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1177(M＋H)＋.MS(ESI)m/z: 1177(M+H) + .

(공정 6) (Process 6)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6- Dihydro-9H-purin-9-yl]-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-14-(6,7,8,9- Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[ 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 5 에서 얻어진 화합물 (828 ㎎) 의 에탄올 (5.0 ㎖) - 테트라하이드로푸란 (5.0 ㎖) 혼합 용액에, 하이드라진 1수화물 (0.342 ㎖) 을 첨가하고, 50 ℃ 에서 6 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (90.9 ㎎ : 불순물 함유) 과 디아스테레오머 2 (91.1 ㎎ : 불순물 함유) 를 얻었다. Hydrazine monohydrate (0.342 ml) was added to a mixed solution of the compound (828 mg) obtained in step 5 in ethanol (5.0 ml) and tetrahydrofuran (5.0 ml), and the mixture was stirred at 50°C for 6 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (90.9 mg: containing impurities) and diastereomer of the title compound. 2 (91.1 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 890(M＋H)＋. MS(ESI) m/z : 890(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 890(M＋H)＋.MS(ESI) m/z : 890(M+H) + .

(공정 7-1) (Process 7-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15-플루오로-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-15-fluoro-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (90.9 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 1) (90.9 mg: containing impurities) obtained in step 6 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 50 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 50% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (20.2 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (20.2 mg).

Figure pat00407
Figure pat00407

(공정 7-2) (Process 7-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15-플루오로-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-15-fluoro-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (91.1 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 2) (91.1 mg: containing impurities) obtained in step 6 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 45% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (26.3 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (26.3 mg).

Figure pat00408
Figure pat00408

실시예 46 : CDN36 의 합성 Example 46: Synthesis of CDN36

N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)-2-하이드록시아세트아미드N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis( Sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H- 5,8-methano- 2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6 -Oxo-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[화학식 184][Formula 184]

Figure pat00409
Figure pat00409

[합성 스킴][Synthesis Scheme]

[화학식 185][Formula 185]

Figure pat00410
Figure pat00410

(공정 1-1) (Process 1-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6 -Oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6, 9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

실시예 45 공정 7-1 에서 얻어진 화합물 (15.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (15.0 mg) obtained in Example 45 Step 7-1, the reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 45% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (10.3 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (10.3 mg).

Figure pat00411
Figure pat00411

(공정 1-2) (Process 1-2)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6 -Oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6, 9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

실시예 45 공정 7-2 에서 얻어진 화합물 (15.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (15.0 mg) obtained in Example 45 Step 7-2, reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 45% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (10.6 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (10.6 mg).

Figure pat00412
Figure pat00412

실시예 47 : CDN37 의 합성 Example 47: Synthesis of CDN37

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15-플루오로-16-하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15-fluoro -16-hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2 -yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa Diphosphacyclotetradecine-2,10-dione

[화학식 186][Formula 186]

Figure pat00413
Figure pat00413

[합성 스킴][Synthesis Scheme]

[화학식 187][Formula 187]

Figure pat00414
Figure pat00414

(공정 1) (Process 1)

2-[(2-아자니우밀에틸)아미노]아데노신 디클로라이드 2-[(2-Azaniumylethyl)amino]adenosine dichloride

이미 알려진 문헌 (WO2012/159072) 의 2-({2-[(tert-부톡시카르보닐)아미노]에틸}아미노)아데노신 (28.7 g) 의 메탄올 (240 ㎖) 용액에, 염화수소의 디옥산 용액 (약 4 M, 240 ㎖) 을 첨가하고, 실온에서 밤새 교반하였다. 반응액을 50 ㎖ 정도까지 감압 농축 후, 석출한 고체를 디에틸에테르로 현탁시켜 여과 채취하고, 표제 화합물 (28.8 g) 을 얻었다. In a methanol (240 ml) solution of 2-({2-[(tert-butoxycarbonyl)amino]ethyl}amino)adenosine (28.7 g) of the previously known literature (WO2012/159072), a dioxane solution of hydrogen chloride ( ca. 4 M, 240 mL) was added and stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to about 50 ml, and the precipitated solid was suspended in diethyl ether and collected by filtration to obtain the title compound (28.8 g).

Figure pat00415
Figure pat00415

(공정 2) (Process 2)

2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 1 에서 얻어진 화합물 (28.8 g) 의 테트라하이드로푸란 (330 ㎖) - 물 (70 ㎖) 혼합액에 트리에틸아민 (37 ㎖) 과 1-({[2-(트리메틸실릴)에톡시]카르보닐}옥시)피롤리딘-2,5-디온 (18.4 g) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액을 감압 농축하고, 톨루엔으로 공비하였다. 잔류물을 톨루엔에 현탁시키고, 고체를 여과 채취하였다. 얻어진 고체를 디클로로메탄과 메탄올에 용해시키고, 감압 농축하였다. 잔류물에 테트라하이드로푸란을 첨가하고, 석출한 고체를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 디클로로메탄과 메탄올에 용해시키고, 감압 농축하였다. 석출한 고체를 디클로로메탄에 현탁시켜 여과 채취하고, 표제 화합물 (22.5 g) 을 얻었다. Triethylamine (37 ml) and 1-({[2-(trimethylsilyl)ethoxy]carbonyl in a mixture of tetrahydrofuran (330 ml)-water (70 ml) of the compound (28.8 g) obtained in step 1 above }Oxy)pyrrolidine-2,5-dione (18.4 g) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and azeotroped with toluene. The residue was suspended in toluene and the solid was collected by filtration. The obtained solid was dissolved in dichloromethane and methanol and concentrated under reduced pressure. Tetrahydrofuran was added to the residue, the precipitated solid was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane and methanol and concentrated under reduced pressure. The precipitated solid was suspended in dichloromethane and collected by filtration to obtain the title compound (22.5 g).

Figure pat00416
Figure pat00416

(공정 3) (Process 3)

N-벤조일-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 2 에서 얻어진 화합물 (24.7 g) 을 사용하여, 실시예 11 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (22.1 g) 을 얻었다. Using the compound (24.7 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 11 step 3 to obtain the title compound (22.1 g).

Figure pat00417
Figure pat00417

(공정 4) (Process 4)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino }Adenosine

상기 공정 3 에서 얻어진 화합물 (22.1 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (29.5 g) 을 얻었다. Using the compound (22.1 g) obtained in the above step 3, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (29.5 g).

Figure pat00418
Figure pat00418

(공정 5) (Process 5)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-{[2-({[2- (trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 4 에서 얻어진 화합물 (29.5 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (6.85 g) 을 얻었다. Using the compound (29.5 g) obtained in the above step 4, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (6.85 g).

Figure pat00419
Figure pat00419

(공정 6) (Process 6)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyano Ethoxy)[di(propan-2-yl)amino]phosphanyl}-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 5 에서 얻어진 화합물 (6.22 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (6.18 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (6.22 g) obtained in step 5 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (6.18 g) was mixed with a mixture of diastereomers on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00420
Figure pat00420

(공정 7) (Process 7)

실시예 44 공정 8 에서 얻어진 화합물 (1.03 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 상기 공정 6 에서 얻어진 화합물 (999 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.03 g) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (999 mg) obtained in step 6 above, a reaction was carried out in the same manner as in step 8 of Example 1, and the obtained crude product was used as it was in the next reaction.

(공정 8) (Process 8)

2-(트리메틸실릴)에틸 [2-({6-벤즈아미드-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]카르바메이트 2-(trimethylsilyl)ethyl [2-({6-benzamide-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2- Cyanoethoxy)-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]carbamate

상기 공정 7 에서 얻어진 혼합물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (370 ㎎ : 불순물 함유) 과 디아스테레오머 2 (201 ㎎ : 불순물 함유) 를 얻었다. Using the mixture obtained in step 7 above, reaction was carried out in the same manner as in Example 1 step 9 to obtain diastereomer 1 (370 mg: impurity contained) and diastereomer 2 (201 mg: impurity contained) of the title compound got

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1307(M-H)-. MS(ESI) m/z : 1307(MH) - .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1307(M-H)-.MS(ESI) m/z : 1307(MH) - .

(공정 9-1) (Process 9-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 1) (370 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (134 ㎎ : 불순물 함유) 을 얻었다. The reaction was carried out in the same manner as in Example 1 Step 10 using the compound (Diastereomer 1) (370 mg: containing impurities) obtained in Step 8 above to obtain the title compound (134 mg: containing impurities).

MS(ESI)m/z : 1048(M＋H)＋.MS(ESI) m/z : 1048(M+H) + .

(공정 9-2) (Process 9-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (디아스테레오머 2) (201 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (49.1 ㎎ : 불순물 함유) 을 얻었다. Using the compound (Diastereomer 2) (201 mg: containing impurities) obtained in Step 8 above, a reaction was carried out in the same manner as in Example 1 Step 10 to obtain the title compound (49.1 mg: containing impurities).

MS(ESI)m/z : 1048(M＋H)＋.MS(ESI) m/z : 1048(M+H) + .

(공정 10-1) (Process 10-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15-플루오로-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15- Fluoro-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 9-1 에서 얻어진 화합물 (134 ㎎) 을 사용하여 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. The compound (134 mg) obtained in step 9-1 was reacted in the same manner as in step 5 of Example 40, and then purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile]. , the title compound was obtained as the triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (22 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (22 mg).

Figure pat00421
Figure pat00421

(공정 10-2) (Process 10-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15-플루오로-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15- Fluoro-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 9-2 에서 얻어진 화합물 (49.1 ㎎ : 불순물 함유) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. After reacting in the same manner as in Example 40 Step 5 using the compound (49.1 mg: containing impurities) obtained in Step 9-2 above, C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] ] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21 mg).

Figure pat00422
Figure pat00422

실시예 48 : CDN38 의 합성 Example 48: Synthesis of CDN38

(5S,7R,8R,12aR,14R,15R,15aS)-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5S,7R,8R,12aR,14R,15R,15aS)-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine-9 -yl] -2,10-bis (sulfanyl) -14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

[화학식 188][Formula 188]

Figure pat00423
Figure pat00423

[합성 스킴][Synthesis Scheme]

[화학식 189][Formula 189]

Figure pat00424
Figure pat00424

(공정 1) (Process 1)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-3'-O-(1H-이미다졸-1-카르보티오일)이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-3'- O-(1H-imidazole-1-carbothioyl)inosine

실시예 22 공정 2 에서 얻어진 화합물 (2'-O-TBS 체) (1.45 g) 의 N,N-디메틸포름아미드 (8.7 ㎖) 용액에, 실온에서 N,N-디메틸-4-아미노피리딘 (213 ㎎) 과 디(1H-이미다졸-1-일)메탄티온 (1.55 g) 을 첨가하고, 20 시간 교반하였다. 반응액을 아세트산에틸로 희석하고, 포화 탄산수소나트륨 수용액과 포화 식염수로 차례로 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.58 g) 을 얻었다.To a solution of the compound obtained in step 2 of Example 22 (2'-O-TBS form) (1.45 g) in N,N-dimethylformamide (8.7 ml), N,N-dimethyl-4-aminopyridine (213 mg) and di(1H-imidazol-1-yl)methanthione (1.55 g) were added, and the mixture was stirred for 20 hours. The reaction solution was diluted with ethyl acetate, and washed sequentially with saturated aqueous sodium hydrogen carbonate solution and saturated brine. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.58 g).

Figure pat00425
Figure pat00425

(공정 2) (Process 2)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-3'-데옥시이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-3'- deoxyinosine

상기 공정 1 에서 얻어진 화합물 (1.69 g) 의 벤젠 (10 ㎖) 용액에, 실온에서 수소화트리부틸주석 (1.42 ㎖) 과 2,2'-아조비스(이소부티로니트릴) (29.4 ㎎) 을 첨가하고, 80 ℃ 에서 4 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.12 g) 을 얻었다. To a solution of the compound (1.69 g) obtained in step 1 above in benzene (10 ml), tributyltin hydride (1.42 ml) and 2,2'-azobis (isobutyronitrile) (29.4 mg) were added at room temperature. , and stirred at 80°C for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.12 g).

Figure pat00426
Figure pat00426

(공정 3) (Process 3)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxyinosine

상기 공정 2 에서 얻어진 화합물 (1.75 g) 의 테트라하이드로푸란 (11 ㎖) 용액에, 0 ℃ 에서 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 2.6 ㎖) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.29 g) 을 얻었다. To a solution of the compound (1.75 g) obtained in step 2 above in tetrahydrofuran (11 ml), a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 2.6 ml) was added at 0°C, followed by stirring at room temperature for 3 hours. did The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.29 g).

Figure pat00427
Figure pat00427

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 4) (Process 4)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-3'-데옥시이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-{(2-cyanoethoxy)[di(propane -2-yl) amino] phosphanyl} -3'-deoxyinosine

상기 공정 3 에서 얻어진 화합물 (1.28 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.47 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다.Using the compound (1.28 g) obtained in step 3 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (1.47 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00428
Figure pat00428

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 836 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (735 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 836 mg). Using the acetonitrile solution of the obtained compound and the compound (735 mg) obtained in the above step 4, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it is for the next reaction.

(공정 6) (Process 6)

2-{9-[(5S,7R,8R,12aR,14R,15R,15aR)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5S,7R,8R,12aR,14R,15R,15aR)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10 -sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (67.6 ㎎) 과 디아스테레오머 2 (91.6 ㎎) 를 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). Using the crude product obtained in step 5 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain diastereomer 1 (67.6 mg) and diastereomer 2 (91.6 mg) of the title compound (HPLC retention time: diastereomer 1 > 2).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1134(M＋H)＋. MS(ESI)m/z: 1134(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1134(M＋H)＋.MS(ESI)m/z: 1134(M+H) + .

(공정 7-1) (Process 7-1)

(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-hydroxyethyl)-6-oxo-1, 6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza Benzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 1) (67.6 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 50 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (34.9 ㎎) 을 얻었다. Using the compound (diastereomer 1) (67.6 mg) obtained in step 6 above, reaction was carried out using the same method as in step 10 of Example 1, and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, Acetonitrile: 0% - 50% (0 min - 30 min)] to obtain the title compound (34.9 mg).

MS(ESI)m/z : 873(M＋H)＋.MS(ESI) m/z : 873(M+H) + .

(공정 7-2) (Process 7-2)

(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-부틸(디메틸)실릴]옥시}-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온 (5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-hydroxyethyl)-6-oxo-1, 6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraaza Benzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 6 에서 얻어진 화합물 (디아스테레오머 2) (91.6 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 50 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (44.2 ㎎) 을 얻었다. Using the compound (diastereomer 2) (91.6 mg) obtained in step 6 above, reaction was carried out using the same method as in step 10 of Example 1, and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, Acetonitrile: 0% - 50% (0 min - 30 min)] to obtain the title compound (44.2 mg).

MS(ESI)m/z : 873(M＋H)＋.MS(ESI) m/z : 873(M+H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5S,7R,8R,12aR,14R,15R,15aS)-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5S,7R,8R,12aR,14R,15R,15aS)-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine -9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7-1 에서 얻어진 화합물 (34.9 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (34.9 mg) obtained in step 7-1 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/acetonitrile]. Purification gave the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21.9 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21.9 mg).

Figure pat00429
Figure pat00429

(공정 8-2) (Process 8-2)

디나트륨 (5S,7R,8R,12aR,14R,15R,15aS)-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5S,7R,8R,12aR,14R,15R,15aS)-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purine -9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7-2 에서 얻어진 화합물 (44.2 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, Sep-Pak (등록상표) C18 [0.1 ％ 트리에틸아민 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (44.2 mg) obtained in step 7-2 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by Sep-Pak (registered trademark) C18 [0.1% aqueous solution of triethylamine/acetonitrile]. Purification gave the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (26.5 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (26.5 mg).

Figure pat00430
Figure pat00430

실시예 49 : CDN39 의 합성 Example 49: Synthesis of CDN39

(5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd ]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 190][Formula 190]

Figure pat00431
Figure pat00431

[합성 스킴][Synthesis Scheme]

[화학식 191][Formula 191]

Figure pat00432
Figure pat00432

(공정 1) (Process 1)

1-[2-(벤조일옥시)에틸]-2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]이노신 1-[2-(benzoyloxy)ethyl]-2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine

시판 (Amadis Chemical) 되는 5'-O-[비스(4-메톡시페닐)(페닐)메틸]이노신 (20.0 g) 의 피리딘 (50 ㎖) - N,N-디메틸아세트아미드 (70.1 ㎖) 혼합 용액에, 4,4'-디메톡시트리틸클로라이드 (12.5 g), 2-브로모에틸벤조에이트 (6.60 ㎖), 및 1,8-디아자비시클로[5.4.0]-7-운데센 (13.1 ㎖) 을 첨가하고, 실온에서 20 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 물을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (24.1 g : 불순물 함유) 을 얻었다. Commercially available (Amadis Chemical) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (20.0 g) pyridine (50 ml) -N,N-dimethylacetamide (70.1 ml) mixed solution E, 4,4'-dimethoxytritylchloride (12.5 g), 2-bromoethylbenzoate (6.60 mL), and 1,8-diazabicyclo[5.4.0]-7-undecene (13.1 mL) ) was added and stirred at room temperature for 20 hours. After stopping the reaction by adding saturated sodium hydrogen carbonate aqueous solution and water to the reaction solution, extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (24.1 g: containing impurities).

MS(ESI)m/z : 1121(M＋H)＋.MS(ESI)m/z: 1121(M+H) + .

(공정 2) (Process 2)

1-[2-(벤조일옥시)에틸]-2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-(트리플루오로메탄술포닐)이노신 1-[2-(benzoyloxy)ethyl]-2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-(trifluoromethanesulfonyl)inosine

상기 공정 1 에서 얻어진 화합물 (24.1 g) 의 디클로로메탄 (120 ㎖) 용액에, 피리딘 (19.0 ㎖) 을 첨가한 후, 0 ℃ 에서 트리플루오로메탄술폰산 무수물 (5.96 ㎖) 을 천천히 적하하고, 동 온도에서 1 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 물을 첨가하여 반응을 정지 후, 디클로로메탄으로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (19.7 g : 불순물 함유) 을 얻었다. To a dichloromethane (120 ml) solution of the compound (24.1 g) obtained in step 1 above, pyridine (19.0 ml) was added, and then trifluoromethanesulfonic anhydride (5.96 ml) was slowly added dropwise at 0°C. was stirred for 1 hour. After stopping the reaction by adding saturated sodium bicarbonate aqueous solution and water to the reaction solution, extraction was performed with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (19.7 g: containing impurities).

MS(ESI)m/z : 1153(M＋H)＋.MS(ESI)m/z: 1153(M+H) + .

(공정 3) (Process 3)

9-{2,5-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-자일로퓨라노실}-1-(2-하이드록시에틸)-1,9-디하이드로-6H-푸린-6-온 9-{2,5-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-xylofuranosyl}-1-(2-hydroxyethyl)-1,9 -dihydro-6H-purin-6-one

상기 공정 2 에서 얻어진 화합물 (19.7 g) 의 N,N-디메틸포름아미드 (85.4 ㎖) 용액에, 아세트산세슘 (8.20 g) 을 첨가하고, 실온에서 16 시간 교반하였다. 반응액에, 메탄올 (85.4 ㎖) 과 탄산칼륨 (4.72 g) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에 물을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (12.2 g : 불순물 함유) 을 얻었다. Cesium acetate (8.20 g) was added to a solution of the compound (19.7 g) obtained in step 2 above in N,N-dimethylformamide (85.4 ml), and the mixture was stirred at room temperature for 16 hours. To the reaction solution, methanol (85.4 ml) and potassium carbonate (4.72 g) were added, and the mixture was stirred at room temperature for 2 hours. After stopping the reaction by adding water to the reaction solution, extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (12.2 g: containing impurities).

MS(ESI)m/z : 917(M＋H)＋.MS(ESI)m/z: 917(M+H) + .

(공정 4) (Process 4)

1-[2-(벤조일옥시)에틸]-9-{2,5-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-자일로퓨라노실}-1,9-디하이드로-6H-푸린-6-온 1-[2-(benzoyloxy)ethyl]-9-{2,5-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-xylofuranosyl}-1 ,9-dihydro-6H-purin-6-one

상기 공정 3 에서 얻어진 화합물 (12.2 g) 의 디클로로메탄 (48.8 ㎖) 용액에, 피리딘 (1.61 ㎖) 과 무수 벤조산 (3.16 g) 을 첨가하고, 실온에서 64 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 물을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (7.36 g : 불순물 함유) 을 얻었다.To a solution of the compound (12.2 g) obtained in step 3 in dichloromethane (48.8 ml), pyridine (1.61 ml) and benzoic acid anhydride (3.16 g) were added, and the mixture was stirred at room temperature for 64 hours. After stopping the reaction by adding saturated sodium hydrogen carbonate aqueous solution and water to the reaction solution, extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (7.36 g: containing impurities).

MS(ESI)m/z : 1021(M＋H)＋.MS(ESI)m/z: 1021(M+H) + .

(공정 5) (Process 5)

1-[2-(벤조일옥시)에틸]-2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-3'-플루오로이노신 1-[2-(benzoyloxy)ethyl]-2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-3'-fluoroinosine

상기 공정 4 에서 얻어진 화합물 (7.36 g) 의 디클로로메탄 (37.0 ㎖) 용액에, 2,6-루티딘 (3.34 ㎖) 을 첨가한 후, -78 ℃ 에서 3불화N,N-디에틸아미노황 (1.42 g) 을 첨가하고, 질소 분위기하, 서서히 실온까지 승온한 후, 밤새 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 천천히 첨가하여 반응을 정지시킨 후, 물을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸 0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (6.89 g : 불순물 함유) 을 얻었다.After adding 2,6-lutidine (3.34 ml) to a dichloromethane (37.0 ml) solution of the compound (7.36 g) obtained in step 4 above, trifluoride N,N-diethylaminosulfur ( 1.42 g) was added, and the temperature was gradually raised to room temperature under a nitrogen atmosphere, followed by stirring overnight. After stopping the reaction by slowly adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, water was added and extraction was performed with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate 0.1% triethylamine] to obtain the title compound (6.89 g: contains impurities).

MS(ESI)m/z : 1023(M＋H)＋.MS(ESI) m/z : 1023(M+H) + .

(공정 6) (Process 6)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-3'-플루오로이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-3'-fluoroinosine

상기 공정 5 에서 얻어진 화합물 (6.89 g) 의 디클로로메탄 (25.0 ㎖) 용액에, 물 (0.303 ㎖) 과 디클로로아세트산 (1.39 ㎖) 을 첨가하고, 실온에서 63 시간 교반하였다. 반응액에 피리딘 (2.0 ㎖) 을 첨가하고, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 및 DIOL 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 조정제하였다. 얻어진 화합물의 피리딘 (25.0 ㎖) 용액에, 0 ℃ 에서 4,4'-디메톡시트리틸클로라이드 (1.83 g) 를 첨가하고, 4 ℃ 에서 21 시간 교반하였다. 반응액에 메탄올 (1.0 ㎖) 을 첨가하고, 4 ℃ 에서 1 시간 교반한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.33 g) 을 얻었다. Water (0.303 ml) and dichloroacetic acid (1.39 ml) were added to a dichloromethane (25.0 ml) solution of the compound (6.89 g) obtained in step 5 above, and the mixture was stirred at room temperature for 63 hours. Pyridine (2.0 ml) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] and DIOL silica gel column chromatography [hexane/ethyl acetate]. To a pyridine (25.0 ml) solution of the obtained compound, 4,4'-dimethoxytrityl chloride (1.83 g) was added at 0°C, and the mixture was stirred at 4°C for 21 hours. Methanol (1.0 ml) was added to the reaction solution, and after stirring at 4°C for 1 hour, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (1.33 g).

Figure pat00433
Figure pat00433

(공정 7) (Process 7)

1-[2-(벤조일옥시)에틸]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-3'-데옥시-3'-플루오로이노신 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-{(2-cyanoethoxy)[di(propane -2-yl) amino] phosphanyl} -3'-deoxy-3'-fluoroinosine

상기 공정 6 에서 얻어진 화합물 (1.33 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.49 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (1.33 g) obtained in step 6 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (1.49 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00434
Figure pat00434

(공정 8) (Process 8)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 948 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 7 에서 얻어진 화합물 (850 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 948 mg). Using the acetonitrile solution of the obtained compound and the compound (850 mg) obtained in the above step 7, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it is for the next reaction.

(공정 9) (Process 9)

2-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-16-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸벤조에이트 2-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-16-fluoro-2-oxo -2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethylbenzoate

상기 공정 8 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (709 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 8 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (709 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1152(M＋H)＋.MS(ESI)m/z: 1152(M+H) + .

(공정 10) (Process 10)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro- 7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (709 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (108 ㎎ : 불순물 함유) 과 디아스테레오머 2 (102 ㎎ : 불순물 함유) 를 얻었다.Using the compound (709 mg) obtained in step 9 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (108 mg: impurity containing) and diastereomer 2 ( 102 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 891(M＋H)＋. MS(ESI) m/z : 891(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 891(M＋H)＋.MS(ESI) m/z : 891(M+H) + .

(공정 11-1) (Process 11-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 10 에서 얻어진 화합물 (디아스테레오머 1) (108 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (108 mg: containing impurity) obtained in Step 10 above, reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 3 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 3% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (40.1 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (40.1 mg).

Figure pat00435
Figure pat00435

(공정 11-2) (Process 11-2)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 10 에서 얻어진 화합물 (디아스테레오머 2) (102 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (102 mg: containing impurities) obtained in Step 10 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 3 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 3% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (26.7 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (26.7 mg).

Figure pat00436
Figure pat00436

실시예 50 : CDN40 의 합성 Example 50: Synthesis of CDN40

(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-16-플루오로-15-하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 16-fluoro-15-hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 192][Formula 192]

Figure pat00437
Figure pat00437

[합성 스킴][Synthesis Scheme]

[화학식 193][Formula 193]

Figure pat00438
Figure pat00438

(공정 1) (Process 1)

3'-데옥시-3'-플루오로이노신 3'-deoxy-3'-fluoroinosine

시판 (Angene) 되는 3'-데옥시-3'-플루오로아데노신 (2.38 g) 의 아세트산 (120 ㎖) 용액에, 아질산나트륨 (6.10 g) 의 수용액 (48 ㎖) 을 조금씩 첨가하고, 실온에서 43 시간 교반하였다. 반응 용액을 감압 농축하고, 톨루엔으로 2 회 공비하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (3.76 g) 을 얻었다. To a commercially available (Angene) solution of 3'-deoxy-3'-fluoroadenosine (2.38 g) in acetic acid (120 ml), an aqueous solution (48 ml) of sodium nitrite (6.10 g) was added little by little, and the mixture was heated to 43 °C at room temperature. Stir for an hour. The reaction solution was concentrated under reduced pressure and azeotroped twice with toluene. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (3.76 g).

Figure pat00439
Figure pat00439

(공정 2) (Process 2)

3'-데옥시-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-3'-플루오로이노신 3'-deoxy-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-3'-fluoroinosine

상기 공정 1 에서 얻어진 화합물 (3.76 g) 의 N,N-디메틸아세트아미드 (37.6 ㎖) 용액에, 2-(2-브로모에틸)-1H-이소인돌-1,3(2H)-디온 (6.60 ㎖) 과 1,8-디아자비시클로[5.4.0]-7-운데센 (3.12 ㎖) 을 첨가하고, 실온에서 3 일간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (3.51 g : 불순물 함유) 을 얻었다.2-(2-bromoethyl)-1H-isoindole-1,3(2H)-dione (6.60 ml) and 1,8-diazabicyclo[5.4.0]-7-undecene (3.12 ml) were added, and the mixture was stirred at room temperature for 3 days. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to obtain the title compound (3.51 g: containing impurities).

Figure pat00440
Figure pat00440

(공정 3) (Process 3)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-3'-플루오로이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindole -2-yl) ethyl] -3'-fluoroinosine

상기 공정 2 에서 얻어진 화합물 (3.51 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.05 g) 을 얻었다. Using the compound (3.51 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (4.05 g).

Figure pat00441
Figure pat00441

(공정 4) (Process 4)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-3'-데옥시-1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-3'-플루오로이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-3 '-Deoxy-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-3'-fluoroinosine

상기 공정 3 에서 얻어진 화합물 (4.05 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.35 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (4.05 g) obtained in step 3 above, a reaction was carried out in the same manner as in step 4 of Example 5, and the title compound (4.35 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00442
Figure pat00442

(공정 5) (Process 5)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 1.47 g). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 4 에서 얻어진 화합물 (1.35 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 1.47 g). Using the acetonitrile solution of the obtained compound and the compound (1.35 g) obtained in the above step 4, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it is for the next reaction.

(공정 6) (Process 6)

3-{[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-7-{1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-16-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-일]옥시}프로판니트릴 3-{[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{1-[2-(1,3-dioxo-1,3-di Hydro-2H-isoindol-2-yl)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-16-fluoro-2-oxo-2-sulfanyl-10- Sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa diphosphacyclotetradecin-10-yl]oxy}propanenitrile

상기 공정 5 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.25 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 5 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (1.25 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1177(M＋H)＋.MS(ESI)m/z: 1177(M+H) + .

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6- Dihydro-9H-purin-9-yl]-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro-2,10-dioxo-14-(6,7,8,9- Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[ 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 6 에서 얻어진 화합물 (1.25 g) 의 에탄올 (7.5 ㎖) - 테트라하이드로푸란 (7.5 ㎖) 혼합 용액에, 하이드라진 1수화물 (0.599 ㎖) 을 첨가하고, 50 ℃ 에서 6 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (145 ㎎ : 불순물 함유) 과 디아스테레오머 2 (198 ㎎ : 불순물 함유) 를 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). Hydrazine monohydrate (0.599 ml) was added to a mixed solution of the compound (1.25 g) obtained in step 6 in ethanol (7.5 ml) and tetrahydrofuran (7.5 ml), and the mixture was stirred at 50°C for 6 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (145 mg: containing impurities) and diastereomer of the title compound. 2 (198 mg: containing impurities) was obtained (retention time by HPLC: diastereomer 1 > 2).

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 890(M＋H)＋. MS(ESI) m/z : 890(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 890(M＋H)＋.MS(ESI) m/z : 890(M+H) + .

(공정 8-1) (Process 8-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-16-fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 1) (145 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (145 mg: containing impurity) obtained in Step 7 above, the reaction was carried out in the same manner as in Step 11 of Example 1, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 5 ％ - 50 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 5% - 50% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (70.7 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (70.7 mg).

Figure pat00443
Figure pat00443

(공정 8-2) (Process 8-2)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-16-fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (198 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (198 mg: containing impurities) obtained in Step 7 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 5 ％ - 50 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 5% - 50% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (69.7 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (69.7 mg).

Figure pat00444
Figure pat00444

실시예 51 : CDN41 의 합성 Example 51: Synthesis of CDN41

N-(2-{9-[(5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-2,10-디옥소-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)-2-하이드록시아세트아미드N-(2-{9-[(5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-2,10-dioxo-2,10-bis( Sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H- 5,8-methano- 2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6 -Oxo-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[화학식 194][Formula 194]

Figure pat00445
Figure pat00445

[합성 스킴][Synthesis Scheme]

[화학식 195][Formula 195]

Figure pat00446
Figure pat00446

(공정 1-1) (Process 1-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6 -Oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6, 9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

실시예 50 공정 8-1 에서 얻어진 화합물 (25.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (25.0 mg) obtained in Example 50 Step 8-1, reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification Conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (18.6 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (18.6 mg).

Figure pat00447
Figure pat00447

(공정 1-2) (Process 1-2)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-플루오로-15-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-16-fluoro-15-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6 -Oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6, 9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

실시예 50 공정 8-2 에서 얻어진 화합물 (디아스테레오머 2) (15.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 2) (15.0 mg) obtained in Example 50 Step 8-2, reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification conditions]; The title compound was obtained as a triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (7.4 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (7.4 mg).

Figure pat00448
Figure pat00448

실시예 52 : CDN42 의 합성 Example 52: Synthesis of CDN42

(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-16-플루오로-15-하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-16-fluoro -15-hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2 -yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa Diphosphacyclotetradecine-2,10-dione

[화학식 196][Formula 196]

Figure pat00449
Figure pat00449

[합성 스킴][Synthesis Scheme]

[화학식 197][Formula 197]

Figure pat00450
Figure pat00450

(공정 1) (Process 1)

2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-2-요오드아데노신 2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-iodoadenosine

시판 (Amadis Chemical) 되는 2-요오드아데노신 (1.35 g) 을 피리딘으로 3 회 공비하였다. 잔류물의 탈수 피리딘 (17.0 ㎖) 용액에, 4,4'-디메톡시트리틸클로라이드 (2.35 g) 를 첨가하고, 질소 분위기, 실온에서 밤새 교반하였다. 반응액에 메탄올 (5.00 ㎖) 을 첨가하여 반응을 정지시킨 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.96 g : 불순물 함유) 을 얻었다.Commercially available (Amadis Chemical) 2-iodoadenosine (1.35 g) was azeotroped with pyridine three times. To a dehydrated pyridine (17.0 ml) solution of the residue, 4,4'-dimethoxytritylchloride (2.35 g) was added, and the mixture was stirred in a nitrogen atmosphere at room temperature overnight. After stopping the reaction by adding methanol (5.00 ml) to the reaction solution, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (1.96 g: containing impurities).

Figure pat00451
Figure pat00451

(공정 2) (Process 2)

2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-2-요오드-3'-O-(트리플루오로메탄술포닐)아데노신2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-iodo-3'-O-(trifluoromethanesulfonyl)adenosine

상기 공정 1 에서 얻어진 화합물 (100 ㎎) 을 톨루엔으로 3 회 공비하였다. 잔류물의 탈수 디클로로메탄 (1.0 ㎖) 용액에, 질소 분위기하, 0 ℃ 에서 피리딘 (0.20 ㎖) 과 트리플루오로메탄술폰산 무수물 (27.0 ㎕) 을 첨가하고, 동 온도에서 80 분간 교반하였다. 트리플루오로메탄술폰산 무수물 (27.0 ㎕) 을 추가하고, 80 분간 더 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (57.2 ㎎) 을 얻었다.The compound (100 mg) obtained in step 1 above was azeotroped with toluene 3 times. To a dehydrated dichloromethane (1.0 ml) solution of the residue, pyridine (0.20 ml) and trifluoromethanesulfonic anhydride (27.0 µl) were added under a nitrogen atmosphere at 0°C, and the mixture was stirred at the same temperature for 80 minutes. Trifluoromethanesulfonic anhydride (27.0 μl) was added and stirred for another 80 minutes. After stopping the reaction by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (57.2 mg).

Figure pat00452
Figure pat00452

(공정 3) (Process 3)

9-{2,5-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-β-D-자일로퓨라노실}-2-요오드-9H-푸린-6-아민 9-{2,5-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-xylofuranosyl}-2-iodo-9H-purin-6-amine

상기 공정 2 에서 얻어진 화합물 (2.76 g) 의 N,N-디메틸포르미아미드 (24.4 ㎖) 용액에 아세트산세슘 (1.20 g) 을 첨가하고, 실온에서 3 시간 교반하였다. 반응액에 메탄올 (24.4 ㎖) 과 탄산칼륨 (675 ㎎) 을 첨가하고, 추가로 2 시간 교반하였다. 반응액에 물을 첨가하여 고체를 석출시킨 후, 감압하에서 메탄올 성분을 증류 제거하였다. 얻어진 고체 여과 채취하여 표제 화합물 (2.38 g) 을 얻었다.Cesium acetate (1.20 g) was added to a solution of the compound (2.76 g) obtained in the above step 2 in N,N-dimethylformiamide (24.4 ml), and the mixture was stirred at room temperature for 3 hours. Methanol (24.4 ml) and potassium carbonate (675 mg) were added to the reaction solution, and the mixture was further stirred for 2 hours. After adding water to the reaction solution to precipitate a solid, the methanol component was distilled off under reduced pressure. The obtained solid was collected by filtration to obtain the title compound (2.38 g).

Figure pat00453
Figure pat00453

(공정 4) (Process 4)

2'5'-비스-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-3'-플루오로-2-요오드아데노신 2'5'-bis-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-3'-fluoro-2-iodoadenosine

상기 공정 3 에서 얻어진 화합물 (2.54 g) 의 디클로로메탄 (17.0 ㎖) 용액에, 피리딘 (1.13 ㎖) 을 첨가한 후, 빙랭하에서 3불화N,N-디에틸아미노황 (0.397 ㎖) 을 첨가하고, 질소 분위기하, 실온에서 40 분간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (2.02 g : 불순물 함유) 을 얻었다. To a solution of the compound (2.54 g) obtained in step 3 above in dichloromethane (17.0 ml), pyridine (1.13 ml) was added, and then N,N-diethylaminosulfur trifluoride (0.397 ml) was added under ice-cooling, The mixture was stirred for 40 minutes at room temperature under a nitrogen atmosphere. After stopping the reaction by adding a saturated aqueous solution of sodium hydrogen carbonate to the reaction solution, extraction was performed with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/0.1% triethylamine] to obtain the title compound (2.02 g: containing impurities).

MS(ESI)m/z : 1000(M＋H)＋.MS(ESI) m/z : 1000(M+H) + .

(공정 5) (Process 5)

3'-데옥시-3'-플루오로-2-요오드아데노신 3'-deoxy-3'-fluoro-2-iodoadenosine

상기 공정 4 에서 얻어진 혼합물 (2.02 g) 의 디클로로메탄 용액 (20.2 ㎖) 에, 빙랭하, 물 (0.364 ㎖) 과 디클로로아세트산 (0.996 ㎖) 을 첨가하고, 실온에서 15 분간 교반하였다. 반응액에 피리딘 (1.95 ㎖) 을 첨가하여 반응을 정지 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.34 g : 불순물 함유) 을 얻었다.To a dichloromethane solution (20.2 ml) of the mixture (2.02 g) obtained in step 4 above, water (0.364 ml) and dichloroacetic acid (0.996 ml) were added under ice-cooling, and the mixture was stirred at room temperature for 15 minutes. After stopping the reaction by adding pyridine (1.95 ml) to the reaction solution, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.34 g: containing impurities).

Figure pat00454
Figure pat00454

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 6) (Process 6)

2-[(2-아미노에틸)아미노]-3'-데옥시-3'-플루오로아데노신 2-[(2-aminoethyl)amino]-3'-deoxy-3'-fluoroadenosine

상기 공정 5 에서 얻어진 화합물 (1.34 g) 에 에틸렌디아민 (1.35 ㎖) 을 첨가하고, 110 ℃ 에서 1 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물 (543 ㎎ : 불순물 함유) 을 얻었다. Ethylenediamine (1.35 ml) was added to the compound (1.34 g) obtained in the above step 5, and the mixture was stirred at 110°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain the title compound (543 mg: containing impurities).

Figure pat00455
Figure pat00455

(공정 7) (Process 7)

3'-데옥시-3'-플루오로-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 3'-deoxy-3'-fluoro-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 6 에서 얻은 화합물 (543 ㎎) 의 테트라하이드로푸란 용액 (10 ㎖) 에, 1-({[2-(트리메틸실릴)에톡시]카르보닐}옥시)피롤리딘-2,5-디온 (961 ㎎), N,N-디메틸포름아미드 (5.0 ㎖), 및 메탄올 (5.0 ㎖) 을 첨가하고, 실온에서 1.5 시간 교반하였다. 1-({[2-(트리메틸실릴)에톡시]카르보닐}옥시)피롤리딘-2,5-디온 (800 ㎎) 을 추가하고, 40 분간 더 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (483 ㎎) 을 얻었다. 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione ( 961 mg), N,N-dimethylformamide (5.0 ml), and methanol (5.0 ml) were added, and the mixture was stirred at room temperature for 1.5 hours. 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (800 mg) was added, and the mixture was further stirred for 40 minutes. After concentration of the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (483 mg).

Figure pat00456
Figure pat00456

(공정 8) (Process 8)

N-벤조일-3'-데옥시-3'-플루오로-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-3'-deoxy-3'-fluoro-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 7 에서 얻은 화합물 (483 ㎎) 을 사용하여, 실시예 11 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (374 ㎎) 을 얻었다.The reaction was carried out in the same manner as in Example 11 Step 3 using the compound (483 mg) obtained in Step 7 above to obtain the title compound (374 mg).

Figure pat00457
Figure pat00457

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 9) (Process 9)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-3'-플루오로-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-3'-fluoro-2-{[2-({[2-(trimethylsilyl) )ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 8 에서 얻어진 화합물 (374 ㎎) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (398 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 11 Step 1 using the compound (374 mg) obtained in Step 8 above to obtain the title compound (398 mg).

Figure pat00458
Figure pat00458

(공정 10) (Process 10)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-3'-데옥시-3'-플루오로-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos panyl}-3'-deoxy-3'-fluoro-2-{[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}adenosine

상기 공정 9 에서 얻어진 화합물 (398 ㎎) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (434 ㎎) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다. Using the compound (398 mg) obtained in step 9 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (434 mg) was added to a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00459
Figure pat00459

(공정 11) (Process 11)

화합물 A : Compound A:

N,N-디에틸에탄아미늄 (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-벤즈아미드-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-16-플루오로-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-티올레이트 N,N-diethylethanelaminium (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-benzamide-2-{[2-({[2-(trimethylsilyl) Ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-16-fluoro-2-oxo-10 -sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecine-2-thiolate

화합물 B : Compound B:

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-벤즈아미드-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-benzamide-2-{[2-({[2-( trimethylsilyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5 ,6-tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro-2,10-dioxoctahydro-2H,10H, 12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10- Bis(thiolate)

실시예 1 공정 7 과 동일한 반응을 이하의 스케일로 실시하였다 (원료 : 502 ㎎). 얻어진 화합물의 아세토니트릴 용액과 상기 공정 10 에서 얻어진 화합물 (434 ㎎) 을 사용하여, 실시예 1 공정 8 및 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 A 의 디아스테레오머 1 (43.9 ㎎) 및 디아스테레오머 2 (27.0 ㎎), 그리고 표제 화합물 B 의 디아스테레오머 1 (55.0 ㎎) 및 디아스테레오머 2 (121 ㎎) 를 얻었다 (각각 불순물 함유). The same reaction as in Step 7 of Example 1 was carried out on the scale shown below (raw material: 502 mg). A reaction was carried out in the same manner as in Example 1 Step 8 and Example 1 Step 9 using the acetonitrile solution of the obtained compound and the compound (434 mg) obtained in Step 10 above to obtain diastereomer 1 of the title compound A ( 43.9 mg) and diastereomer 2 (27.0 mg), and diastereomer 1 (55.0 mg) and diastereomer 2 (121 mg) of the title compound B (each containing impurities).

화합물 A 의 디아스테레오머 1 (저극성) Diastereomer 1 of Compound A (low polarity)

MS(ESI)m/z : 1309(M＋H)＋. MS(ESI)m/z: 1309(M+H) + .

화합물 A 의 디아스테레오머 2 (고극성) Diastereomer 2 of compound A (high polarity)

MS(ESI)m/z : 1309(M＋H)＋. MS(ESI)m/z: 1309(M+H) + .

화합물 B 의 디아스테레오머 1 (저극성) Diastereomer 1 of compound B (low polarity)

MS(ESI)m/z : 1256(M＋H)＋. MS(ESI) m/z : 1256(M+H) + .

화합물 B 의 디아스테레오머 2 (고극성) Diastereomer 2 of compound B (high polarity)

MS(ESI)m/z : 1256(M＋H)＋.MS(ESI) m/z : 1256(M+H) + .

(공정 12-1) (Process 12-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 11 에서 얻어진 화합물 A (디아스테레오머 1) (43.9 ㎎) 를 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 얻었다.The reaction was carried out in the same manner as in Example 1 Step 10 using Compound A (Diastereomer 1) (43.9 mg) obtained in Step 11 above to obtain the title compound.

MS(ESI)m/z : 1048(M＋H)＋.MS(ESI) m/z : 1048(M+H) + .

(공정 12'-1) (Process 12'-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 11 에서 얻어진 화합물 B (디아스테레오머 1) (55.0 ㎎) 를 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 얻었다.The reaction was carried out in the same manner as in Example 1 Step 10 using Compound B (Diastereomer 1) (55.0 mg) obtained in Step 11 above to obtain the title compound.

MS(ESI)m/z : 1048(M＋H)＋.MS(ESI) m/z : 1048(M+H) + .

(공정 12-2) (Process 12-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-15-{[tert-부틸(디메틸)실릴]옥시}-16-플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-16-fluoro-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8 -Methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 11 에서 얻어진 화합물 A (디아스테레오머 2) (27.0 ㎎ : 불순물 함유) 를 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물을 얻었다. The reaction was carried out in the same manner as in Example 1 Step 10 using Compound A (Diastereomer 2) (27.0 mg: containing impurities) obtained in Step 11 above to obtain the title compound.

MS(ESI)m/z : 1048(M＋H)＋.MS(ESI) m/z : 1048(M+H) + .

(공정 13-1) (Process 13-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-16-fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 - Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 12-1 및 12'-1 에서 얻어진 화합물을 아울러, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (36.4 ㎎) 을 얻었다. The compounds obtained in steps 12-1 and 12'-1 were reacted together in the same manner as in step 11 in Example 1 to obtain the title compound (36.4 mg).

MS(ESI)m/z : 934(M＋H)＋.MS(ESI) m/z : 934(M+H) + .

(공정 13-2) (Process 13-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-16-fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 - Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 12-2 에서 얻어진 화합물을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (12.4 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 1 step 11 using the compound obtained in step 12-2 to obtain the title compound (12.4 mg).

MS(ESI)m/z : 934(M＋H)＋.MS(ESI) m/z : 934(M+H) + .

(공정 14-1) (Process 14-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-16- Fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 13-1 에서 얻어진 화합물 (36.4 ㎎) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. The compound (36.4 mg) obtained in step 13-1 was reacted in the same manner as in step 5 of Example 40, and then purified under the following [purification conditions] to obtain the title compound as a triethylamine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 40 분)] 및 Sep-Pak (등록상표) C18 [물/아세토니트릴/0.1 ％ 트리에틸아민]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 40 min)] and Sep-Pak (registered trademark) C18 [water/acetonitrile/0.1 % triethylamine].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21.0 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21.0 mg).

Figure pat00460
Figure pat00460

(공정 14-2) (Process 14-2)

비스(N,N,N-트리부틸부탄-1-아미늄) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N,N-tributylbutan-1-aminium) (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl )amino]-9H-purin-9-yl}-16-fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3, 5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 13-2 에서 얻어진 화합물 (29.5 ㎎) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물 (24.2 ㎎ : 불순물 함유) 을 얻었다. Using the compound (29.5 mg) obtained in step 13-2 above, the reaction was carried out in the same manner as in step 5 of Example 40, and then purified under the following [purification conditions] to obtain the title compound (24.2 mg: containing impurities). got it

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)], Sep-Pak (등록상표) C18 [물/아세토니트릴/0.1 ％ 트리에틸아민], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 25 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)], Sep-Pak (registered trademark) C18 [water/acetonitrile/0.1 % triethylamine], and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 25% (0 min - 30 min)].

Figure pat00461
Figure pat00461

(공정 14-2') (Process 14-2')

디나트륨 (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-16-플루오로-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-16- Fluoro-15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 14-2 에서 얻어진 화합물 (19.0 ㎎ : 불순물 함유) 을, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 25 ％ (0 분 - 30 분)] 로 정제하였다.The compound obtained in step 14-2 (19.0 mg: containing impurities) was purified by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 25% (0 min - 30 min)] .

얻어진 화합물을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (8.4 ㎎) 을 얻었다.The obtained compound was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (8.4 mg).

Figure pat00462
Figure pat00462

실시예 53 : CDN43 의 합성 Example 53: Synthesis of CDN43

(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-아미노-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15-하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15 -Hydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

[화학식 198][Formula 198]

Figure pat00463
Figure pat00463

[합성 스킴][Synthesis Scheme]

[화학식 199][Formula 199]

Figure pat00464
Figure pat00464

(공정 1) (Process 1)

2'-O-아세틸-3'-아지드-5'-O-벤조일-3'-데옥시-N-(2-메틸프로파노일)구아노신 2'-O-acetyl-3'-azide-5'-O-benzoyl-3'-deoxy-N-(2-methylpropanoyl)guanosine

이미 알려진 문헌 (Recl. Trav. Chim. Pay-Bas 1986, 105, 85-91) 의 1,2-디-O-아세틸-3-아지드-5-O-벤조일-3-데옥시-D-리보퓨라노스 (4.0 g) 의 아세토니트릴 (60 ㎖) 용액에, N2-이소부티릴구아닌 (3.65 g) 과 N,O-비스(트리메틸실릴)아세트아미드 (8.08 ㎖) 를 실온에서 첨가하고, 70 ℃ 에서 3 시간 교반하였다. 트리메틸실릴트리플루오로메탄술포네이트 (2.98 ㎖) 를 70 ℃ 에서 첨가하고, 동 온도에서 1 일간 교반하였다. 반응액을 냉각 후, 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시키고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (4.82 g) 을 얻었다.1,2-di-O-acetyl-3-azide-5-O-benzoyl-3-deoxy-D- of the previously known literature (Recl. Trav. Chim. Pay-Bas 1986, 105, 85-91) To a solution of ribofuranose (4.0 g) in acetonitrile (60 ml), N2-isobutyrylguanine (3.65 g) and N,O-bis(trimethylsilyl)acetamide (8.08 ml) were added at room temperature and It was stirred at °C for 3 hours. Trimethylsilyltrifluoromethanesulfonate (2.98 ml) was added at 70°C, and the mixture was stirred at the same temperature for 1 day. After cooling the reaction solution, a saturated aqueous solution of sodium hydrogen carbonate was added to stop the reaction, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (4.82 g).

Figure pat00465
Figure pat00465

(공정 2) (Process 2)

3'-아지드-3'-데옥시-N-(2-메틸프로파노일)구아노신 3'-Azide-3'-deoxy-N-(2-methylpropanoyl)guanosine

상기 공정 1 에서 얻어진 화합물 (5.48 g) 의 테트라하이드로푸란 (64 ㎖) - 메탄올 (32 ㎖) 혼합 용액에, 5 M 수산화나트륨 (17 ㎖) 을 0 ℃ 에서 첨가하고, 15 분간 교반하였다. 반응액에 아세트산 (5.08 ㎖) 을 첨가하여 반응을 정지시키고, 반응액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (3.8 g) 을 얻었다. To a mixed solution of the compound (5.48 g) obtained in step 1 above in tetrahydrofuran (64 ml) and methanol (32 ml) was added 5 M sodium hydroxide (17 ml) at 0°C, and the mixture was stirred for 15 minutes. Acetic acid (5.08 ml) was added to the reaction mixture to stop the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (3.8 g).

Figure pat00466
Figure pat00466

(공정 3) (Process 3)

3'-아지드-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-N-(2-메틸프로파노일)구아노신 3'-Azide-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-N-(2-methylpropanoyl)guanosine

상기 공정 2 에서 얻어진 화합물 (3.8 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (5.78 g) 을 얻었다. Using the compound (3.8 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 11 step 1 to obtain the title compound (5.78 g).

Figure pat00467
Figure pat00467

(공정 4) (Process 4)

3'-아미노-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-N-(2-메틸프로파노일)구아노신 3'-Amino-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-N-(2-methylpropanoyl)guanosine

상기 공정 3 에서 얻어진 화합물 (5.17 g) 의 메탄올 (60 ㎖) 용액에, 트리페닐포스핀 (3.98 g) 을 첨가하고, 실온에서 4 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.22 g) 을 얻었다. Triphenylphosphine (3.98 g) was added to a solution of the compound (5.17 g) obtained in step 3 in methanol (60 ml), and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (2.22 g).

Figure pat00468
Figure pat00468

(공정 5) (Process 5)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-데옥시-N-(2-메틸프로파노일)-3'-(2,2,2-트리플루오로아세트아미드)구아노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-deoxy-N-(2-methylpropanoyl)-3'-(2,2,2-trifluoro acetamide) guanosine

상기 공정 4 에서 얻어진 화합물 (2.22 g) 의 N,N-디메틸포름아미드 (20 ㎖) 용액에, 트리플루오로아세트산에틸 (4.0 ㎖) 을 첨가하고, 50 ℃ 에서 2 일간 교반하였다. 반응액을 냉각 후, 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시키고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (2.06 g) 을 얻었다. Ethyl trifluoroacetate (4.0 ml) was added to a solution of the compound (2.22 g) obtained in step 4 in N,N-dimethylformamide (20 ml), and the mixture was stirred at 50°C for 2 days. After cooling the reaction solution, a saturated aqueous solution of sodium hydrogen carbonate was added to stop the reaction, followed by extraction with ethyl acetate. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (2.06 g).

Figure pat00469
Figure pat00469

(공정 6) (Process 6)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-3'-데옥시-N-(2-메틸프로파노일)-3'-(2,2,2-트리플루오로아세트아미드)구아노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-3 '-Deoxy-N-(2-methylpropanoyl)-3'-(2,2,2-trifluoroacetamide)guanosine

상기 공정 5 에서 얻어진 화합물 (1.0 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (780 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다.Using the compound (1.0 g) obtained in step 5 above, a reaction was carried out in the same manner as in step 4 of Example 5 to obtain the title compound (780 mg) as a mixture of diastereomers on a phosphorus atom.

(공정 7) (Process 7)

2-메틸프로판-2-아미늄 6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3-O-[옥사이드(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 2-methylpropane-2-aminium 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[oxide(oxo)-λ 5 -phosphanyl]-β-D -ribofuranosyl} -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

실시예 1 공정 6 에서 얻어진 화합물 (5.0 g) 의 아세토니트릴 (30 ㎖) 용액에, 물 (0.18 ㎖) 과 트리플루오로아세트산피리딘염 (1.2 g) 을 첨가하고, 실온에서 30 분간 교반하였다. 반응액에 tert-부틸아민 (30 ㎖) 을 첨가하고, 실온에서 30 분간 교반하였다. 반응액을 감압 농축 후, 잔류물을 아세토니트릴로 2 회 공비하였다. 잔류물의 디클로로메탄 (50 ㎖) 용액에 물 (0.88 ㎖) 과 디클로로아세트산 (3.2 ㎖) 의 디클로로메탄 (50 ㎖) 용액을 차례로 첨가하고, 실온에서 1 시간 교반하였다. 메탄올 (5 ㎖) 과 피리딘 (6.3 ㎖) 을 첨가하여 반응을 정지시킨 후, 반응액을 감압 농축하였다. 잔류물을 DIOL 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸 → 디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (3.0 g) 을 얻었다.Example 1 To a solution of the compound (5.0 g) obtained in Step 6 in acetonitrile (30 ml), water (0.18 ml) and trifluoroacetic acid pyridine salt (1.2 g) were added, and the mixture was stirred at room temperature for 30 minutes. To the reaction solution was added tert-butylamine (30 ml), and the mixture was stirred at room temperature for 30 minutes. After the reaction solution was concentrated under reduced pressure, the residue was azeotroped twice with acetonitrile. To a dichloromethane (50 ml) solution of the residue was added successively a dichloromethane (50 ml) solution of water (0.88 ml) and dichloroacetic acid (3.2 ml), and the mixture was stirred at room temperature for 1 hour. After stopping the reaction by adding methanol (5 ml) and pyridine (6.3 ml), the reaction solution was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [hexane/ethyl acetate→dichloromethane/methanol] to obtain the title compound (3.0 g).

Figure pat00470
Figure pat00470

(공정 8) (Process 8)

디뉴클레오티드 A dinucleotide A

상기 공정 7 에서 얻어진 화합물 (543 ㎎) 의 디클로로메탄 (6.0 ㎖) - 아세토니트릴 (6.0 ㎖) 혼합 용액에, 몰레큘러 시브스 3A, 1/16 (500 ㎎) 과 4,5-디시아노이미다졸 (126 ㎎) 을 첨가하고, 실온에서 15 분간 교반하였다. 반응액에 상기 공정 6 에서 얻어진 화합물 (780 ㎎) 을 첨가하고, 5 시간 교반 후, N,N-디메틸-N'-(3-술파닐리덴-3H-1,2,4-디티아졸-5-일)메탄이미드아미드 (219 ㎎) 를 첨가하고, 추가로 2 시간 교반하였다. 반응액으로부터 몰레큘러 시브스 3A 를 여과 제거하고, 여과액에 포화 탄산수소나트륨 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하였다. 얻어진 화합물 (740 ㎎) 의 디클로로메탄 (6.0 ㎖) 용액에, 물 (0.086 ㎖) 과 디클로로아세트산 (0.158 ㎖) 의 디클로로메탄 (6.0 ㎖) 용액을 차례로 첨가하고, 실온에서 1.5 시간 교반하였다. 피리딘 (0.31 ㎖) 을 첨가하여 반응을 정지 후, 반응액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (520 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. To a mixed solution of dichloromethane (6.0 ml) - acetonitrile (6.0 ml) of the compound (543 mg) obtained in step 7 above, molecular sieves 3A, 1/16 (500 mg) and 4,5-dicyanoimidazole (126 mg) was added and stirred at room temperature for 15 minutes. To the reaction mixture was added the compound (780 mg) obtained in step 6, and after stirring for 5 hours, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazole-5 -1) Methanimideamide (219 mg) was added, and the mixture was further stirred for 2 hours. Molecular Sieves 3A was removed by filtration from the reaction solution, and a saturated aqueous solution of sodium hydrogencarbonate was added to the filtrate, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate]. To a solution of the obtained compound (740 mg) in dichloromethane (6.0 ml), water (0.086 ml) and a dichloromethane (6.0 ml) solution of dichloroacetic acid (0.158 ml) were added in this order, and the mixture was stirred at room temperature for 1.5 hours. After stopping the reaction by adding pyridine (0.31 ml), the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (520 mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z : 1168(M＋H)＋.MS(ESI)m/z: 1168(M+H) + .

(공정 9) (Process 9)

N-{9-[(5S,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴-16-(2,2,2-트리플루오로아세트아미드)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-2-일}-2-메틸프로판아미드 N-{9-[(5S,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl -10-sulfanylidene-16-(2,2,2-trifluoroacetamide)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}- 2-methylpropanamide

상기 공정 8 에서 얻어진 화합물 (270 ㎎) 을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (110 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the compound (270 mg) obtained in step 8 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (110 mg) as a diastereomer mixture on a phosphorus atom.

MS(ESI)m/z : 1182(M＋H)＋.MS(ESI) m/z : 1182(M+H) + .

(공정 10) (Process 10)

비스(N,N-디에틸에탄아미늄) (5S,7R,8R,12aR,14R,15R,15aR,16R)-16-아미노-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15-{[tert-부틸(디메틸)실릴]옥시}-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5S,7R,8R,12aR,14R,15R,15aR,16R)-16-amino-7-(2-amino-6-oxo-1,6-di hydro-9H-purin-9-yl)-15-{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2 ,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (110 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (34.2 ㎎) 과 디아스테레오머 2 (19.3 ㎎) 를 얻었다. Using the compound (110 mg) obtained in step 9 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (34.2 mg) and diastereomer 2 (19.3 mg) of the title compound. got

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 859(M＋H)＋. MS(ESI) m/z : 859(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 859(M＋H)＋.MS(ESI) m/z : 859(M+H) + .

(공정 11-1) (Process 11-1)

디나트륨 (5S,7R,8R,12aR,14R,15R,15aS,16R)-16-아미노-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 10 에서 얻어진 화합물 (디아스테레오머 1) (34.2 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 1) (34.2 mg) obtained in step 10 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetate]. nitrile] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (8.9 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (8.9 mg).

Figure pat00471
Figure pat00471

(공정 11-2) (Process 11-2)

디나트륨 (5S,7R,8R,12aR,14R,15R,15aS,16R)-16-아미노-7-(2-아미노-6-옥소-1,6-디하이드로-9H-푸린-9-일)-15-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -15-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 10 에서 얻어진 화합물 (디아스테레오머 2) (19.3 ㎎) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 2) (19.3 mg) obtained in step 10 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetate]. nitrile] to obtain the title compound as a triethylamine salt.

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (5.6 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (5.6 mg).

Figure pat00472
Figure pat00472

실시예 54 : CDN44 의 합성 Example 54: Synthesis of CDN44

(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-디플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro- 9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 200][Formula 200]

Figure pat00473
Figure pat00473

[합성 스킴][Synthesis Scheme]

[화학식 201][Formula 201]

Figure pat00474
Figure pat00474

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (636 ㎎) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 49 공정 7 에서 얻어진 화합물 (640 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (636 mg) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (640 mg) obtained in Example 49 Step 7, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-10-(2-시아노에톡시)-15,16-디플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸벤조에이트 2-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-10-(2-cyanoethoxy)-15,16-difluoro-2-oxo-2-sulfanyl-10-sulfanylideneocta hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyn-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethylbenzoate

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (228 ㎎ : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 1 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (228 mg: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1040(M＋H)＋.MS(ESI) m/z : 1040(M+H) + .

(공정 3) (Process 3)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-디플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-7-[1-(2-hydroxyethyl)-6-oxo-1,6-di hydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

상기 공정 2 에서 얻어진 화합물 (228 ㎎) 을 메탄올 (5 ㎖) 과 28 ％ 암모니아 수용액 (5 ㎖) 에 용해하고, 60 ℃ 에서 12 시간 교반하였다. 반응액을 감압 농축 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 30 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 7 ％ - 50 ％ (0 분 - 40 분)] 로 차례로 정제하고, 표제 화합물의 디아스테레오머 1 과 디아스테레오머 2 를 각각 트리에틸아민염으로서 얻었다 (HPLC 의 유지 시간 : 디아스테레오머 1 ＞ 2). The compound (228 mg) obtained in the above step 2 was dissolved in methanol (5 ml) and 28% aqueous ammonia (5 ml), and the mixture was stirred at 60°C for 12 hours. After concentration of the reaction solution under reduced pressure, C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution/acetonitrile, acetonitrile: 5% to 30% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 7% - 50% (0 min - 40 min)] to obtain diastereomer 1 and diastereomer 2 of the title compound as triethylamine salts, respectively (retention time of HPLC: diastereomer 1 > 2).

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물의 디아스테레오머 1 (12.5 ㎎) 과 디아스테레오머 2 (15.8 ㎎) 를 얻었다. The obtained triethylamine salt was subjected to salt exchange in the same manner as [Conversion to sodium salt] described in Step 11 of Example 1 to obtain diastereomer 1 (12.5 mg) and diastereomer 2 (15.8 mg) of the title compound. got

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00475
Figure pat00475

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00476
Figure pat00476

실시예 55 : CDN45 의 합성 Example 55: Synthesis of CDN45

(5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디플루오로-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16- Difluoro-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-dione

[화학식 202][Formula 202]

Figure pat00477
Figure pat00477

[합성 스킴][Synthesis Scheme]

[화학식 203][Formula 203]

Figure pat00478
Figure pat00478

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (696 ㎎) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 52 공정 10 에서 얻어진 화합물 (738 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (696 mg) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (738 mg) obtained in Example 52 Step 10, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-(트리메틸실릴)에틸 [2-({6-벤즈아미드-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-10-(2-시아노에톡시)-15,16-디플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]카르바메이트 2-(trimethylsilyl)ethyl [2-({6-benzamide-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) -10- (2-cyanoethoxy) -15,16-difluoro-2 -Oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6, 9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]carbamate

상기 공정 1 에서 얻어진 혼합물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물을 포함하는 혼합물 (1.31 g) 을 얻었다. 얻어진 혼합물은 그대로 다음의 반응에 사용하였다. Using the mixture obtained in step 1 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain a mixture (1.31 g) containing the title compound. The obtained mixture was used as it was for the next reaction.

MS(ESI)m/z : 1195(M-H)-.MS(ESI) m/z : 1195(MH) - .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-15,16-디플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-15,16-difluoro-2,10-dioxo-14-(6,7,8,9- Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[ 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 2 에서 얻어진 혼합물 (1.31 g) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (249 ㎎ : 불순물 함유) 과 디아스테레오머 2 (344 ㎎ : 불순물 함유) 를 각각 얻었다. Using the mixture (1.31 g) obtained in step 2 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (249 mg: containing impurities) and diastereomer 2 (344 mg) of the title compound. : containing impurities) were respectively obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 936(M＋H)＋. MS(ESI) m/z : 936(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 936(M＋H)＋.MS(ESI) m/z : 936(M+H) + .

(공정 4-1) (Step 4-1)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15, 16-difluoro-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 1) (249 ㎎ : 불순물 함유) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (249 mg: containing impurities) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 40 Step 5, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 40 ％ (0 분 - 30 분)] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 0 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 40% (0 min - 30 min)] and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile Nitrile: 0% - 30% (0 min - 30 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (9.2 ㎎) 을 얻었다. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (9.2 mg).

Figure pat00479
Figure pat00479

(공정 4-2) (Process 4-2)

디나트륨 (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-15,16-디플루오로-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8S,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15, 16-difluoro-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo Tetradecyne-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 2) (344 ㎎ : 불순물 포함한다) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (344 mg: containing impurities) obtained in Step 3 above, reaction was carried out in the same manner as in Example 40 Step 5, and then purified under the following [Purification conditions] to obtain the title compound was obtained as a triethylamine salt.

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 40 ％ (0 분 - 30 분)] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 10% - 40% (0 min - 30 min)] and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile Nitrile: 10% - 30% (0 min - 30 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (4.7 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (4.7 mg).

Figure pat00480
Figure pat00480

실시예 56 : CDN46 의 합성 Example 56: Synthesis of CDN46

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)데카하이드로-2H,10H-5,8-메타노-2λ5,10λ5-시클로펜타[l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) )-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)decahydro-2H,10H-5,8- Methano-2λ 5 ,10λ 5 -cyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 204][Formula 204]

Figure pat00481
Figure pat00481

[합성 스킴][Synthesis Scheme]

[화학식 205][Formula 205]

Figure pat00482
Figure pat00482

(공정 1) (Process 1)

(1R,2S,3R,5R)-3-(4-클로로-5-요오드-7H-피롤로[2,3-d]피리미딘-7-일)-5-(하이드록시메틸)시클로펜탄-1,2-디올 (1R,2S,3R,5R)-3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)cyclopentane- 1,2-diols

이미 알려진 문헌 (WO2015/199136) 의 7-[(3aS,4R,6R,6aR)-6-({[tert-부틸(디메틸)실릴]옥시}메틸)-2,2-디메틸테트라하이드로-2H,3aH-시클로펜타[d][1,3]디옥솔-4-일]-4-클로로-5-요오드-7H-피롤로[2,3-d]피리미딘 (6.0 g) 에, 트리플루오로아세트산 (36 ㎖) 과 물 (12 ㎖) 을 차례로 첨가하고, 실온에서 3 시간 교반하였다. 반응액을 감압 농축하고, 표제 화합물의 미정제체를 얻었다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.7-[(3aS,4R,6R,6aR)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2,2-dimethyltetrahydro-2H of the known literature (WO2015/199136), 3aH-cyclopenta[d][1,3]dioxol-4-yl]-4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (6.0 g) in trifluoro Acetic acid (36 ml) and water (12 ml) were added in that order, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product of the title compound. The obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

(1R,2S,3R,5R)-3-(4-아미노-5-요오드-7H-피롤로[2,3-d]피리미딘-7-일)-5-(하이드록시메틸)시클로펜탄-1,2-디올 (1R,2S,3R,5R)-3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)cyclopentane- 1,2-diols

상기 공정 1 에서 얻어진 화합물 (4.4 g) 의 1,4-디옥산 (40 ㎖) 용액에, 28 ％ 암모니아수 (40 ㎖) 를 첨가하고, 90 ℃ 에서 72 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [물/메탄올] 로 정제하고, 표제 화합물 (3.4 g) 을 얻었다. 28% aqueous ammonia (40 ml) was added to a solution of the compound (4.4 g) obtained in step 1 in 1,4-dioxane (40 ml), and the mixture was stirred at 90°C for 72 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [water/methanol] to obtain the title compound (3.4 g).

Figure pat00483
Figure pat00483

(공정 3) (Process 3)

7-[(4aR,6R,7S,7aR)-2,2-디-tert-부틸-7-{[tert-부틸(디메틸)실릴]옥시}헥사하이드로-2H-시클로펜타[d][1,3,2]디옥사실린-6-일]-5-요오드-7H-피롤로[2,3-d]피리미딘-4-아민 7-[(4aR,6R,7S,7aR)-2,2-di-tert-butyl-7-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-2H-cyclopenta[d][1, 3,2]dioxacillin-6-yl]-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine

상기 공정 2 에서 얻어진 화합물 (3.3 g) 을 사용하여, 실시예 1 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (4.6 g) 을 얻었다. Using the compound (3.3 g) obtained in the above step 2, a reaction was carried out in the same manner as in Example 1 step 1 to obtain the title compound (4.6 g).

Figure pat00484
Figure pat00484

(공정 4) (Process 4)

7-[(4aR,6R,7S,7aR)-2,2-디-tert-부틸-7-{[tert-부틸(디메틸)실릴]옥시}헥사하이드로-2H-시클로펜타[d][1,3,2]디옥사실린-6-일]-5-(3,3-디에톡시프로파-1-인-1-일)-7H-피롤로[2,3-d]피리미딘-4-아민 7-[(4aR,6R,7S,7aR)-2,2-di-tert-butyl-7-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-2H-cyclopenta[d][1, 3,2]dioxacillin-6-yl]-5-(3,3-diethoxypropan-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-4- amine

상기 공정 3 에서 얻어진 화합물 (4.6 g) 을 사용하여, 실시예 1 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (3.6 g) 을 얻었다.Using the compound (4.6 g) obtained in the above step 3, a reaction was carried out in the same manner as in Example 1 step 2 to obtain the title compound (3.6 g).

Figure pat00485
Figure pat00485

(공정 5) (Process 5)

2-[(4aR,6R,7S,7aR)-2,2-디-tert-부틸-7-{[tert-부틸(디메틸)실릴]옥시}헥사하이드로-2H-시클로펜타[d][1,3,2]디옥사실린-6-일]-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 2-[(4aR,6R,7S,7aR)-2,2-di-tert-butyl-7-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-2H-cyclopenta[d][1, 3,2]dioxacillin-6-yl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 4 에서 얻어진 화합물 (3.60 g) 을 사용하여, 실시예 1 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.44 g) 을 얻었다. Using the compound (3.60 g) obtained in the above step 4, a reaction was carried out in the same manner as in Example 1 step 3 to obtain the title compound (2.44 g).

Figure pat00486
Figure pat00486

(공정 6) (Process 6)

{2-[(4aR,6R,7S,7aR)-2,2-디-tert-부틸-7-{[tert-부틸(디메틸)실릴]옥시}헥사하이드로-2H-시클로펜타[d][1,3,2]디옥사실린-6-일]-2,7,8,9-테트라하이드로-6H-2,3,5,6-테트라아자벤조[cd]아줄렌-6-일}(페닐)메타논 {2-[(4aR,6R,7S,7aR)-2,2-di-tert-butyl-7-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-2H-cyclopenta[d][1 ,3,2]dioxacillin-6-yl] -2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo [cd] azulen-6-yl} (phenyl )methanone

상기 공정 5 에서 얻어진 화합물 (2.44 g) 을 사용하여, 실시예 1 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.01 g) 을 얻었다. Using the compound (2.44 g) obtained in the above step 5, a reaction was carried out in the same manner as in Example 1 step 4 to obtain the title compound (2.01 g).

Figure pat00487
Figure pat00487

(공정 7) (Process 7)

{2-[(1R,2S,3R,4R)-4-{[비스(4-메톡시페닐)(페닐)메톡시]메틸}-2-{[tert-부틸(디메틸)실릴]옥시}-3-하이드록시시클로펜틸]-2,7,8,9-테트라하이드로-6H-2,3,5,6-테트라아자벤조[cd]아줄렌-6-일}(페닐)메타논 {2-[(1R,2S,3R,4R)-4-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-2-{[tert-butyl(dimethyl)silyl]oxy}- 3-hydroxycyclopentyl]-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulen-6-yl}(phenyl)methanone

상기 공정 6 에서 얻어진 화합물 (2.01 g) 을 사용하여, 실시예 1 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.13 g) 을 얻었다.Using the compound (2.01 g) obtained in the above step 6, a reaction was carried out in the same manner as in Example 1 step 5 to obtain the title compound (2.13 g).

Figure pat00488
Figure pat00488

(공정 8) (Process 8)

(1R,2S,3R,5R)-3-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-5-{[비스(4-메톡시페닐)(페닐)메톡시]메틸}-2-{[tert-부틸(디메틸)실릴]옥시}시클로펜틸 4-옥소펜타노에이트 (1R,2S,3R,5R)-3-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-2-{[tert-butyl(dimethyl)silyl]oxy}cyclopentyl 4-oxopentanoate

레불린산 (2.96 g) 의 테트로하이드로푸란 (20 ㎖) 용액에, N,N-디시클로헥실카르보디이미드 (2.63 g) 를 첨가하고, 실온에서 12 시간 교반하였다. 석출물을 여과 제거한 후, 여과액을 감압 농축하였다. 잔류물의 디클로로메탄 (20 ㎖) 용액에, 상기 공정 7 에서 얻어진 화합물 (2.10 g) 과 4-디메틸아미노피리딘 (155 ㎎) 을 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지 후, 아세트산에틸로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.35 g) 을 얻었다. To a solution of levulinic acid (2.96 g) in tetrahydrofuran (20 ml), N,N-dicyclohexylcarbodiimide (2.63 g) was added, and the mixture was stirred at room temperature for 12 hours. After filtering off the precipitate, the filtrate was concentrated under reduced pressure. To a dichloromethane (20 ml) solution of the residue, the compound (2.10 g) obtained in step 7 and 4-dimethylaminopyridine (155 mg) were added, and the mixture was stirred at room temperature for 1 hour. After stopping the reaction by adding a saturated aqueous solution of sodium hydrogencarbonate to the reaction solution, extraction was performed with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (2.35 g).

Figure pat00489
Figure pat00489

(공정 9) (Process 9)

(1R,2S,3R,5R)-3-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-2-{[tert-부틸(디메틸)실릴]옥시}-5-(하이드록시메틸)시클로펜틸-4-옥소펜타노에이트 (1R,2S,3R,5R)-3-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl )-2-{[tert-butyl(dimethyl)silyl]oxy}-5-(hydroxymethyl)cyclopentyl-4-oxopentanoate

상기 공정 8 에서 얻어진 화합물 (2.35 g) 의 디클로로메탄 (25 ㎖) 용액에, 물 (0.24 ㎖) 과 디클로로아세트산 (1.05 ㎖) 의 디클로로메탄 (25 ㎖) 용액을 차례로 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 메탄올 (1.0 ㎖) 과 포화 탄산수소나트륨 수용액을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.15 g) 을 얻었다. To a dichloromethane (25 ml) solution of the compound (2.35 g) obtained in step 8 above, water (0.24 ml) and a dichloromethane (25 ml) solution of dichloroacetic acid (1.05 ml) were sequentially added, and the mixture was stirred at room temperature for 1 hour. did After stopping the reaction by adding methanol (1.0 ml) and saturated sodium hydrogencarbonate aqueous solution to the reaction solution, extraction was performed with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.15 g).

Figure pat00490
Figure pat00490

(공정 10) (Process 10)

N-벤조일-2'-O-[({(1R,2R,3S,4R)-4-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-3-{[tert-부틸(디메틸)실릴]옥시}-2-[(4-옥소펜타노일)옥시]시클로펜틸}메톡시)(2-시아노에톡시)포스포로티오일]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]아데노신 N-benzoyl-2'-O-[({(1R,2R,3S,4R)-4-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-3-{[tert-butyl(dimethyl)silyl]oxy}-2-[(4-oxopentanoyl)oxy]cyclopentyl}methoxy)(2- Cyanoethoxy)phosphorothioyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]adenosine

상기 공정 9 에서 얻어진 화합물 (550 ㎎) 의 아세토니트릴 (12 ㎖) 용액에, 몰레큘러 시브스 3A, 1/16 (500 ㎎) 과 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (1.23 g) 을 첨가하고, 실온에서 15 분 교반하였다. 반응액에 4,5-디시아노이미다졸을 첨가하여 1 시간 교반 후, 3H-1,2-벤조티올-3-온 1,1-디옥사이드 (355 ㎎) 를 첨가하고, 추가로 1 시간 교반하였다. 반응액으로부터 몰레큘러 시브스 3A 를 여과 제거하고, 여과액에 포화 탄산수소나트륨 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.17 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. To a solution of the compound (550 mg) obtained in step 9 in acetonitrile (12 ml), molecular sieves 3A, 1/16 (500 mg) and N-benzoyl-5'-O-[bis(4-methoxy Phenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl } Adenosine (1.23 g) was added and stirred at room temperature for 15 minutes. After adding 4,5-dicyanoimidazole to the reaction solution and stirring for 1 hour, 3H-1,2-benzothiol-3-one 1,1-dioxide (355 mg) was added, followed by further stirring for 1 hour. . Molecular Sieves 3A was removed by filtration from the reaction solution, and a saturated aqueous solution of sodium hydrogencarbonate was added to the filtrate, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (1.17 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1539(M＋H)＋.MS(ESI)m/z: 1539(M+H) + .

(공정 11) (Process 11)

N-벤조일-2'-O-[{[(1R,2R,3S,4R)-4-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-3-{[tert-부틸(디메틸)실릴]옥시}-2-하이드록시시클로펜틸]메톡시}(2-시아노에톡시)포스포로티오일]-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]아데노신 N-benzoyl-2'-O-[{[(1R,2R,3S,4R)-4-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-3-{[tert-butyl(dimethyl)silyl]oxy}-2-hydroxycyclopentyl]methoxy}(2-cyanoethoxy)phosphorothiocyanate Oil]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]adenosine

상기 공정 10 에서 얻어진 화합물 (1.22 g) 의 아세토니트릴 (2.0 ㎖) 용액에, 하이드라진 1수화물 (0.25 ㎖) 의 아세트산 (5.0 ㎖) - 피리딘 (7.5 ㎖) 혼합 용액을 첨가하고, 실온에서 30 분 교반하였다. 반응액에 물을 첨가하여 반응을 정지시킨 후, 아세트산에틸로 추출하고, 유기층을 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (770 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. A mixed solution of hydrazine monohydrate (0.25 ml) in acetic acid (5.0 ml) - pyridine (7.5 ml) was added to a solution of the compound (1.22 g) obtained in step 10 in acetonitrile (2.0 ml), and the mixture was stirred at room temperature for 30 minutes. did After stopping the reaction by adding water to the reaction solution, extraction was performed with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (770 mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z : 1442(M＋H)＋.MS(ESI) m/z : 1442(M+H) + .

(공정 12) (Process 12)

N-벤조일-2'-O-[{[(1R,2R,3S,4R)-4-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-3-{[tert-부틸(디메틸)실릴]옥시}-2-{[하이드록시(옥소)-λ5-포스파닐]옥시}시클로펜틸]메톡시}(2-시아노에톡시)포스포로티오일]-3'-O-[tert-부틸(디메틸)실릴]아데노신 N-benzoyl-2'-O-[{[(1R,2R,3S,4R)-4-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6- tetraazabenzo[cd]azulen-2-yl)-3-{[tert-butyl(dimethyl)silyl]oxy}-2-{[hydroxy(oxo)-λ 5 -phosphanyl]oxy}cyclopentyl] Methoxy}(2-cyanoethoxy)phosphorothioyl]-3'-O-[tert-butyl(dimethyl)silyl]adenosine

상기 공정 11 에서 얻어진 화합물 (770 ㎎) 의 피리딘 (8.0 ㎖) 용액에, 아인산디페닐 (0.61 ㎖) 을 첨가하고, 실온에서 2 시간 교반하였다. 반응액에 물 (20 ㎖), 아세토니트릴 (8.0 ㎖), 및 아세트산트리에틸암모늄 수용액 (2 M, 1.6 ㎖) 을 첨가하고, 1 시간 교반 후, 반응액을 감압 농축하였다. 잔류물의 디클로로메탄 (5.0 ㎖) 용액에, 물 (0.096 ㎖) 과 디클로로아세트산 (0.22 ㎖) 의 디클로로메탄 (5.0 ㎖) 용액을 차례로 첨가하고, 30 분간 교반하였다. 피리딘 (0.43 ㎖) 을 첨가하여 반응을 정지시킨 후, 반응액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (400 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Diphenyl phosphite (0.61 ml) was added to a solution of the compound (770 mg) obtained in step 11 in pyridine (8.0 ml), and the mixture was stirred at room temperature for 2 hours. Water (20 ml), acetonitrile (8.0 ml), and an aqueous solution of triethylammonium acetate (2 M, 1.6 ml) were added to the reaction solution, and after stirring for 1 hour, the reaction solution was concentrated under reduced pressure. To a dichloromethane (5.0 ml) solution of the residue, water (0.096 ml) and a dichloromethane (5.0 ml) solution of dichloroacetic acid (0.22 ml) were added in that order, and the mixture was stirred for 30 minutes. After stopping the reaction by adding pyridine (0.43 ml), the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (400 mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z : 1203(M＋H)＋.MS(ESI) m/z : 1203(M+H) + .

(공정 13) (Process 13)

N-{9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-2-옥소-2-술파닐-10-술파닐리덴데카하이드로-2H,10H-5,8-메타노-2λ5,10λ5-시클로펜타[l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2 -sulfanyl-10-sulfanilidenedecahydro-2H,10H-5,8-methano-2λ 5 ,10λ 5 -cyclopenta[l][1,3,6,9,11,2,10]penta Oxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 12 에서 얻어진 화합물 (400 ㎎) 을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (300 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the compound (400 mg) obtained in step 12 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (300 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1217(M＋H)＋.MS(ESI)m/z: 1217(M+H) + .

(공정 14) (Process 14)

디나트륨 (5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)데카하이드로-2H,10H-5,8-메타노-2λ5,10λ5-시클로펜타[l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo -14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)decahydro-2H,10H-5,8-meta No-2λ 5 ,10λ 5 -Cyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 13 에서 얻어진 화합물 (300 ㎎) 의 메탄올 (10 ㎖) 용액에, 28 ％ 암모니아수 (10 ㎖) 를 첨가하고, 실온에서 12 시간 교반하였다. 반응액을 감압 농축 후, 잔류물에 트리에틸아민3불화수소산염 (3.0 ㎖) 을 첨가하고, 45 ℃ 에서 2 시간 교반하였다. 빙랭한 1 M 탄산수소트리에틸암모늄 용액 (18 ㎖) - 트리에틸아민 (6.0 ㎖) 혼합 용액에 반응액을 적하하여 반응을 정지시켰다. 반응액을 감압 농축 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 과 디아스테레오머 2 를 각각 트리에틸아민염으로서 얻었다. 28% aqueous ammonia (10 ml) was added to a solution of the compound (300 mg) obtained in step 13 in methanol (10 ml), and the mixture was stirred at room temperature for 12 hours. After concentrating the reaction solution under reduced pressure, triethylamine hydrofluoric acid salt (3.0 ml) was added to the residue, and the mixture was stirred at 45°C for 2 hours. The reaction solution was added dropwise to an ice-cooled 1 M triethylammonium hydrogen carbonate solution (18 ml) -triethylamine (6.0 ml) mixed solution to stop the reaction. The reaction solution was concentrated under reduced pressure and purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 and diastereomer 2 of the title compound as triethylamine salts, respectively.

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물의 디아스테레오머 1 (45.6 ㎎) 과 디아스테레오머 2 (12.6 ㎎) 를 얻었다.The obtained triethylamine salt was subjected to salt exchange by the same method as [Conversion to sodium salt] described in Step 11 of Example 1 to obtain diastereomer 1 (45.6 mg) and diastereomer 2 (12.6 mg) of the title compound. got

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

Figure pat00491
Figure pat00491

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

Figure pat00492
Figure pat00492

실시예 57 : CDN47 의 합성 Example 57: Synthesis of CDN47

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2,10-비스(술파닐)-10-술파닐리덴-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-온(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl) ) -10-sulfanylidene-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulen-2-yl) octahydro-2H, 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2- on

[화학식 206][Formula 206]

Figure pat00493
Figure pat00493

[합성 스킴][Synthesis Scheme]

[화학식 207][Formula 207]

Figure pat00494
Figure pat00494

(공정 1) (Process 1)

6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-O-[tert-부틸(디메틸)실릴]-3-O-(4-옥소펜타노일)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-(4-oxopentanoyl )-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

실시예 1 공정 5 에서 얻어진 화합물을 사용하여, 실시예 56 공정 8 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.8 g) 을 얻었다.Using the compound obtained in Example 1 Step 5, reaction was carried out in the same manner as in Example 56 Step 8 to obtain the title compound (2.8 g).

Figure pat00495
Figure pat00495

(공정 2) (Process 2)

6-벤조일-2-{2-O-[tert-부틸(디메틸)실릴]-3-O-(4-옥소펜타노일)-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-(4-oxopentanoyl)-β-D-ribofuranosyl}-6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 1 에서 얻어진 화합물 (2.8 g) 을 사용하여, 실시예 56 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.74 g) 을 얻었다.Using the compound (2.8 g) obtained in the above step 1, a reaction was carried out in the same manner as in Example 56 step 9 to obtain the title compound (1.74 g).

Figure pat00496
Figure pat00496

(공정 3) (Process 3)

N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-[(2-시아노에톡시){[(2,4-디클로로페닐)메틸]술파닐}포스포로티오일]아데노신 N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[(2-cyano ethoxy){[(2,4-dichlorophenyl)methyl]sulfanyl}phosphorothioyl]adenosine

시판 (ChemGenes) 되는 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}아데노신 (3.73 g) 의 아세토니트릴 (30 ㎖) 용액에, 몰레큘러 시브스 3A, 1/16 (1.0 g) 과 2,4-디클로로벤질메르캅탄 (1.8 ㎖) 을 첨가하고, 실온에서 10 분간 교반하였다. 반응액에 이미다졸과염소산염 (3.18 g) 을 첨가하여 2.5 시간 교반 후, 황 (242 ㎎) 을 첨가하고, 추가로 1 시간 교반하였다. 반응액으로부터 몰레큘러 시브스 3A 를 여과 제거하고, 여과액에 포화 탄산수소나트륨 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.73 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다.Commercially available (ChemGenes) N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{ To a solution of (2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (3.73 g) in acetonitrile (30 mL), Molecular Sieves 3A, 1/16 (1.0 g) and 2,4-dichlorobenzylmercaptan (1.8 ml) were added, and the mixture was stirred at room temperature for 10 minutes. After adding imidazole perchlorate (3.18 g) to the reaction solution and stirring for 2.5 hours, sulfur (242 mg) was added and the mixture was further stirred for 1 hour. Molecular Sieves 3A was removed by filtration from the reaction solution, and a saturated aqueous solution of sodium hydrogencarbonate was added to the filtrate, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (2.73 g) as a mixture of diastereomers on a phosphorus atom.

Figure pat00497
Figure pat00497

(공정 4) (Process 4)

N,N-디에틸에탄아미늄 N-벤조일-5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-[{[(2,4-디클로로페닐)메틸]술파닐}(술파이드)포스포릴]아데노신 N,N-diethylethanelaminium N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2' -O-[{[(2,4-dichlorophenyl)methyl]sulfanyl}(sulfide)phosphoryl]adenosine

상기 공정 3 에서 얻어진 화합물 (2.66 g) 의 아세토니트릴 (30 ㎖) 용액에, 트리에틸아민 (30 ㎖) 을 첨가하고, 실온에서 4 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 DIOL 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.5 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다.Triethylamine (30 ml) was added to a solution of the compound (2.66 g) obtained in step 3 in acetonitrile (30 ml), and the mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by DIOL silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (2.5 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1058(M＋H)＋.MS(ESI)m/z: 1058(M+H) + .

(공정 5) (Process 5)

디뉴클레오티드 B dinucleotide B

상기 공정 4 에서 얻어진 화합물 (2.5 g) 의 디클로로메탄 (10 ㎖) 용액에, 몰레큘러 시브스 4A, 1/16 (1.0 g), 상기 공정 2 에서 얻어진 화합물 (930 ㎎), 및 1-메틸이미다졸 (1.18 ㎖) 을 차례로 첨가하고, 실온에서 20 분간 교반하였다. 반응액에 2,4,6-트리이소프로필벤젠술포닐클로라이드 (905 ㎎) 를 첨가하고, 추가로 4 시간 교반하였다. 반응액으로부터 몰레큘러 시브스 4A 를 여과 제거하고, 여과액에 포화 탄산수소나트륨 수용액을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.04 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. To a dichloromethane (10 ml) solution of the compound (2.5 g) obtained in step 4 above, molecular sieves 4A, 1/16 (1.0 g), the compound obtained in step 2 (930 mg), and 1-methyl Midazole (1.18 mL) was added one after another and stirred at room temperature for 20 minutes. 2,4,6-triisopropylbenzenesulfonyl chloride (905 mg) was added to the reaction mixture, and the mixture was further stirred for 4 hours. Molecular Sieves 4A was removed by filtration from the reaction solution, and a saturated aqueous solution of sodium hydrogencarbonate was added to the filtrate, followed by extraction with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to give the title compound (1.04 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1662(M＋H)＋.MS(ESI) m/z : 1662(M+H) + .

(공정 6) (Process 6)

디뉴클레오티드 C dinucleotide C

상기 공정 5 에서 얻어진 화합물 (1.04 g) 을 사용하여, 실시예 56 공정 11 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (820 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the compound (1.04 g) obtained in step 5 above, a reaction was carried out in the same manner as in step 11 of Example 56 to obtain the title compound (820 mg) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1564(M＋H)＋.MS(ESI)m/z: 1564(M+H) + .

(공정 7) (Process 7)

디뉴클레오티드 D dinucleotide D

상기 공정 6 에서 얻어진 화합물 (820 ㎎) 을 사용하여, 실시예 56 공정 12 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (440 ㎎) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다.Using the compound (820 mg) obtained in Step 6 above, a reaction was carried out in the same manner as in Example 56 Step 12 to obtain the title compound (440 mg) as a mixture of diastereomers on a phosphorus atom.

(공정 8) (Process 8)

N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-10-{[(2,4-디클로로페닐)메틸]술파닐}-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}벤즈아미드 N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5, 6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-{[(2,4-dichlorophenyl)methyl]sulfanyl }-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3 ,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide

상기 공정 7 에서 얻어진 화합물 (440 ㎎) 을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (360 ㎎) 을 얻었다. Using the compound (440 mg) obtained in step 7 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (360 mg).

MS(ESI)m/z : 1340(M＋H)＋.MS(ESI) m/z : 1340(M+H) + .

(공정 9) (Process 9)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-벤즈아미드-9H-푸린-9-일)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-benzamide-9H-purin-9-yl)-14-( 6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl( dimethyl)silyl]oxy}-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3 ,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (360 ㎎) 의 디메틸술폭시드 용액에, 1-도데칸티올 (434 ㎎) 과 1,8-디아자비시클로[5.4.0]-7-운데센 (0.40 ㎖) 을 첨가하고, 실온에서 6 시간 교반하였다. 반응액을 그대로 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물 (130 ㎎) 을 얻었다. To a dimethylsulfoxide solution of the compound (360 mg) obtained in step 8 above, 1-dodecanethiol (434 mg) and 1,8-diazabicyclo[5.4.0]-7-undecene (0.40 ml) were added. and stirred at room temperature for 6 hours. The reaction mixture was purified as it was by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain the title compound (130 mg).

MS(ESI)m/z : 1182(M＋H)＋.MS(ESI) m/z : 1182(M+H) + .

(공정 10) (Process 10)

디나트륨 (2S,5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-아미노-9H-푸린-9-일)-15,16-디하이드록시-2-옥소-10-술파닐리덴-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (2S,5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2-oxo- 10-sulfanylidene-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H; 12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10- Bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (130 ㎎) 을 사용하여, 실시예 56 공정 14 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (130 mg) obtained in step 9 above, reaction was carried out in the same manner as in step 14 of Example 56, followed by purification by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile], The title compound was obtained as a triethylamine salt.

얻어진 트리에틸아민염을 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (62.8 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Example 1 Step 11 to obtain the title compound (62.8 mg).

Figure pat00498
Figure pat00498

실시예 58 : CDN48 의 합성 Example 58: Synthesis of CDN48

(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2(7H )-yl)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2, 10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 208][Formula 208]

Figure pat00499
Figure pat00499

[합성 스킴][Synthesis Scheme]

[화학식 209][Formula 209]

Figure pat00500
Figure pat00500

(공정 1) (Process 1)

4-클로로-5-요오드-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘 4-chloro-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

시판 (PharmaBlock) 되는 4-클로로-5-요오드-7H-피롤로[2,3-d]피리미딘 (73.8 g) 의 N,N-디메틸포르미아미드 (10 ㎖) 용액에, 빙랭하에서 수소화나트륨 (미네랄 오일을 45 ％ 함유) (13.3 g) 을 첨가하고, 실온까지 승온하면서 40 분간 교반하였다. 재차 빙랭하고, [2-(클로로메톡시)에틸](트리메틸)실란 (51.0 ㎖) 을 10 분간 걸쳐 첨가한 후, 동 온도에서 30 분간 교반하였다. 대부분이 고화한 반응액에 물 (260 ㎖) 을 조금씩 첨가하여 반응을 정지시켰다. 고체를 여과 채취하고, 물 (1500 ㎖) 과 헥산 (600 ㎖) 으로 세정 후, 감압하 40 ℃ 에서 건조시키고, 표제 화합물 (97.63 g) 을 얻었다. To a solution of commercially available (PharmaBlock) 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (73.8 g) in N,N-dimethylformiamide (10 ml), sodium hydride was added under ice-cooling. (containing 45% of mineral oil) (13.3 g) was added, and the mixture was stirred for 40 minutes while raising the temperature to room temperature. After ice-cooling again, and [2-(chloromethoxy)ethyl](trimethyl)silane (51.0 ml) was added over 10 minutes, it stirred at the same temperature for 30 minutes. Water (260 ml) was added little by little to the mostly solidified reaction liquid to stop the reaction. The solid was collected by filtration, washed with water (1500 mL) and hexane (600 mL), and dried at 40°C under reduced pressure to obtain the title compound (97.63 g).

Figure pat00501
Figure pat00501

(공정 2) (Process 2)

4-(벤질옥시)-5-요오드-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘4-(Benzyloxy)-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

벤질알코올 (27 ㎖) 의 N,N-디메틸포르미아미드 (170 ㎖) 용액에, 빙랭하에서 수소화나트륨 (미네랄 오일을 45 ％ 함유) (12 g) 을 첨가하고, 실온까지 승온하면서 40 분간 교반하였다. 재차 빙랭하고, 상기 공정 1 에서 얻어진 화합물 (97.63 g) 의 N,N-디메틸포르미아미드 (360 ㎖) 현탁액을 40 분간 걸쳐 첨가하고, 동 온도에서 35 분간 교반하였다. 반응액에 빙편 (氷片) 과 포화 염화암모늄 수용액을 첨가하여 반응을 정지시켰다. 반응액을 포화 염화암모늄 수용액과 아세트산에틸의 2 층에 따르고, 아세트산에틸 : 톨루엔 (9 : 1) 으로 추출하였다. 유기층을 물과 포화 식염수로 2 회씩 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (107.7 g) 을 얻었다. Sodium hydride (containing 45% mineral oil) (12 g) was added to a solution of benzyl alcohol (27 ml) in N,N-dimethylformiamide (170 ml) under ice-cooling, and the mixture was stirred for 40 minutes while heating to room temperature. . After ice-cooling again, a suspension of the compound (97.63 g) obtained in step 1 above in N,N-dimethylformiamide (360 ml) was added over 40 minutes, followed by stirring at the same temperature for 35 minutes. The reaction was stopped by adding ice cubes and a saturated aqueous ammonium chloride solution to the reaction solution. The reaction solution was poured into two layers of saturated aqueous ammonium chloride and ethyl acetate, and extracted with ethyl acetate:toluene (9:1). The organic layer was washed twice each with water and brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (107.7 g).

Figure pat00502
Figure pat00502

(공정 3) (Process 3)

4-(벤질옥시)-5-(3,3-디에톡시프로파-1-인-1-일)-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘 4-(benzyloxy)-5-(3,3-diethoxyprop-1-yn-1-yl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2 ,3-d]pyrimidine

상기 공정 2 에서 얻어진 화합물 (113.4 g) 의 아세토니트릴 (1000 ㎖) - 트리에틸아민 (98 ㎖) 혼합 용액에, 질소 분위기하, 실온에서 요오드화구리 (4.49 g), 테트라키스트리페닐포스핀팔라듐(0) (8.17 g), 및 3,3-디에톡시프로파-1-인 (104 ㎖) 을 첨가하고, 동 온도에서 4.5 시간 교반하였다. 반응액을 감압 농축 후, 잔류물에 아세트산에틸과 헥산을 첨가하고, 석출한 고체를 여과 제거하였다. 고체를 아세트산에틸 : 헥산 (1 : 1) 의 혼합액으로 세정 후, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (145.5 g : 불순물 함유) 을 얻었다. Copper iodide (4.49 g), tetrakistriphenylphosphine palladium ( 0) (8.17 g) and 3,3-diethoxypropa-1-yne (104 ml) were added, and the mixture was stirred at the same temperature for 4.5 hours. After concentration of the reaction solution under reduced pressure, ethyl acetate and hexane were added to the residue, and the precipitated solid was filtered off. The solid was washed with a mixture of ethyl acetate and hexane (1:1), and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (145.5 g: contains impurities).

MS(ESI)m/z : 482(M＋H)＋.MS(ESI) m/z : 482(M+H) + .

(공정 4) (Process 4)

5-(3,3-디에톡시프로필)-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘-4-올 5-(3,3-diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-ol

상기 공정 3 에서 얻어진 화합물 (145.5 g) 의 에탄올 (900 ㎖) 용액에, 10 ％ 팔라듐탄소 촉매 (M) wet (50.2 g) 를 첨가하고, 수소 분위기하, 실온에서 5 시간 교반하였다. 반응액에 디클로로메탄 (500 ㎖) 을 첨가하고, 셀라이트로 촉매를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 2 회 정제하고, 표제 화합물 (59.6 g) 을 얻었다.A 10% palladium carbon catalyst (M) wet (50.2 g) was added to a solution of the compound (145.5 g) obtained in step 3 in ethanol (900 ml), and the mixture was stirred at room temperature under a hydrogen atmosphere for 5 hours. Dichloromethane (500 ml) was added to the reaction mixture, the catalyst was filtered off through celite, and the filtrate was concentrated under reduced pressure. The residue was purified twice by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (59.6 g).

Figure pat00503
Figure pat00503

(공정 5) (Process 5)

5-(3,3-디에톡시프로필)-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘-4-티올 5-(3,3-diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine-4-thiol

상기 공정 4 에서 얻어진 화합물 (59.6 g) 의 탈수 디클로로메탄 (300 ㎖) 용액에, 질소 분위기하, 2,6-루티딘 (42 ㎖) 을 첨가하였다. 트리플루오로메탄술폰산 무수물 (31 ㎖) 을 -20 ℃ 에서 20 분간 걸쳐 적하하고, 동 온도에서 20 분간 교반하였다. N,N-디메틸포름아미드 (500 ㎖) 와 1황화수소나트륨 n 수화물 (33.5 g) 을 빙랭하에서 첨가하고, 실온까지 승온한 후, 2.5 시간 교반하였다. 반응액을 감압 농축하고, 저비점 성분을 증류 제거하였다. 잔류물을 아세트산에틸과 빙랭한 포화 염화암모늄 수용액의 2 층에 따르고, 아세트산에틸 : 톨루엔 (9 : 1) 의 혼합액으로 추출하였다. 유기층을 포화 염화암모늄 수용액으로 1 회, 포화 식염수로 2 회 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물과 2,6-루티딘의 혼합물을 얻었다. 얻어진 혼합물을 아세트산에틸과 1 규정 염산의 2 층에 따르고, 아세트산에틸로 2 회 추출하였다. 유기층을 포화 식염수로 3 회 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하고, 표제 화합물 (57.6 g) 을 얻었다.To a dehydrated dichloromethane (300 ml) solution of the compound (59.6 g) obtained in step 4 above, 2,6-lutidine (42 ml) was added under a nitrogen atmosphere. Trifluoromethanesulfonic anhydride (31 ml) was added dropwise over 20 minutes at -20°C, and stirred at the same temperature for 20 minutes. After adding N,N-dimethylformamide (500 ml) and sodium hydrogen sulfide n hydrate (33.5 g) under ice-cooling, and raising the temperature to room temperature, it stirred for 2.5 hours. The reaction solution was concentrated under reduced pressure, and the low boiling point components were distilled off. The residue was poured into two layers of ethyl acetate and ice-cooled saturated aqueous ammonium chloride solution, and extracted with a mixture of ethyl acetate and toluene (9:1). The organic layer was washed once with saturated aqueous ammonium chloride solution and twice with saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain a mixture of the title compound and 2,6-lutidine. The resulting mixture was poured into two layers of ethyl acetate and 1N hydrochloric acid, and extracted twice with ethyl acetate. The organic layer was washed 3 times with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure to obtain the title compound (57.6 g).

Figure pat00504
Figure pat00504

(공정 6) (Process 6)

3-(4-술파닐-7-{[2-(트리메틸실릴)에톡시]메틸}-7H-피롤로[2,3-d]피리미딘-5-일)프로판-1-올 3-(4-sulfanyl-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propan-1-ol

상기 공정 5 에서 얻어진 화합물 (31.62 g) 을 80 ％ 아세트산 수용액 (300 ㎖) 에 용해하고, 실온에서 30 분간 교반하였다. 원료의 소실을 확인 후, 빙랭하고, 수소화붕소나트륨 (1.45 g) 을 소량씩 주의 깊게 첨가하고, 동 온도에서 30 분간 교반하였다. 이어서, 수소화트리아세톡시붕소나트륨 (24.4 g) 을 15 분간 걸쳐 첨가하고, 동 온도에서 1.5 시간 교반하였다. 반응액을 5 분의 1 정도의 양까지 감압 농축하였다. 잔류물에 탄산수소나트륨 (고체) 을 주의 깊게 첨가하여 어느 정도 중화한 후, 아세트산에틸로 추출하였다. 유기층을 포화 탄산수소나트륨과 포화 식염수로 차례로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (17.93 g) 을 얻었다. The compound (31.62 g) obtained in the above step 5 was dissolved in an 80% aqueous acetic acid solution (300 ml), and the mixture was stirred at room temperature for 30 minutes. After confirming that the raw material had disappeared, it was ice-cooled, and sodium borohydride (1.45 g) was carefully added in small portions, followed by stirring at the same temperature for 30 minutes. Subsequently, sodium triacetoxyboron hydride (24.4 g) was added over 15 minutes, and the mixture was stirred at the same temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure to about 1/5 of the amount. Sodium hydrogen carbonate (solid) was carefully added to the residue to neutralize it to some extent, followed by extraction with ethyl acetate. The organic layer was washed sequentially with saturated sodium hydrogen carbonate and brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (17.93 g).

Figure pat00505
Figure pat00505

(공정 7) (Process 7)

2-{[2-(트리메틸실릴)에톡시]메틸}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-{[2-(trimethylsilyl)ethoxy]methyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 6 에서 얻어진 화합물 (31.31 g) 의 탈수 테트라하이드로푸란 (600 ㎖) 용액에, 질소 분위기하, 0 ℃ 에서 트리페닐포스핀 (25.4 g) 과 디이소프로필아조디카르복실레이트 (21.8 g) 를 첨가하고, 동 온도에서 1 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/아세트산에틸] 및 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 차례로 정제하고, 표제 화합물 (35.93 g : 불순물 함유) 을 얻었다. Triphenylphosphine (25.4 g) and diisopropyl azodicarboxylate (21.8 g) were added to a solution of the compound (31.31 g) obtained in step 6 above in dehydrated tetrahydrofuran (600 ml) at 0°C under a nitrogen atmosphere. was added and stirred at the same temperature for 1 hour. After concentration of the reaction solution under reduced pressure, the residue was purified sequentially by silica gel column chromatography [dichloromethane/ethyl acetate] and silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (35.93 g: contains impurities). .

Figure pat00506
Figure pat00506

(공정 8) (Process 8)

(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)메탄올 (8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)methanol

상기 공정 7 에서 얻어진 화합물 (35.93 g) 의 디클로로메탄 (150 ㎖) 용액에, 실온에서 트리플루오로아세트산 (150 ㎖) 을 첨가하고, 동 온도에서 1.5 시간 교반하였다. 반응액을 감압 농축 후, 톨루엔으로 4 회 공비하였다. 잔류물에 디클로로메탄 : 헥산 (1 : 2) 의 혼합액을 첨가하고, 석출한 고체를 여과 채취하였다 (고체 1). 여과액을 감압 농축 후, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸 → 아세트산에틸/메탄올] 로 정제하고, 고체 2 를 얻었다. 고체 1 과 고체 2 를 아울러, 표제 화합물 (20.13 g) 을 얻었다. To a dichloromethane (150 ml) solution of the compound (35.93 g) obtained in step 7 above, trifluoroacetic acid (150 ml) was added at room temperature, and the mixture was stirred at the same temperature for 1.5 hours. After the reaction solution was concentrated under reduced pressure, it was azeotroped with toluene 4 times. A mixed solution of dichloromethane:hexane (1:2) was added to the residue, and the precipitated solid was collected by filtration (solid 1). After concentration of the filtrate under reduced pressure, the residue was purified by silica gel column chromatography [hexane/ethyl acetate→ethyl acetate/methanol] to obtain a solid 2. Solid 1 and Solid 2 were combined to obtain the title compound (20.13 g).

Figure pat00507
Figure pat00507

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 9) (Process 9)

2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 8 에서 얻어진 화합물 (20.13 g) 의 메탄올 (250 ㎖) 현탁액에, 28 ％ 암모니아 수용액 (150 ㎖) 을 첨가하고, 실온에서 1.5 시간 교반하였다. 반응액을 감압하에서 절반 정도의 양까지 농축하였다. 석출한 고체를 여과 채취하고, 에탄올로 세정하고, 고체 1 을 얻었다. 여과액을 감압 농축하고, 동일한 조작으로 고체 2 를 얻었다. 여과액을 실리카 겔에 바른 후, 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축하고, 에탄올로 슬러리 세정 후, 고체를 여과 채취하였다 (고체 3). 고체 1, 고체 2, 및 고체 3 을 아울러, 표제 화합물 (12.36 g) 을 얻었다.A 28% aqueous ammonia solution (150 ml) was added to a methanol (250 ml) suspension of the compound (20.13 g) obtained in step 8, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated to about half the amount under reduced pressure. The precipitated solid was collected by filtration and washed with ethanol to obtain Solid 1. The filtrate was concentrated under reduced pressure, and solid 2 was obtained by the same operation. After applying the filtrate to silica gel, it was purified by silica gel column chromatography [dichloromethane/methanol]. A fraction containing the target substance was concentrated under reduced pressure, and the solid was collected by filtration after washing the slurry with ethanol (solid 3). Solid 1, Solid 2, and Solid 3 were combined to obtain the title compound (12.36 g).

Figure pat00508
Figure pat00508

(공정 10) (Process 10)

2-(2,3,5-트리-O-벤질-β-D-아라비노퓨라노실)-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-(2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo [cd] Azulene

상기 공정 9 에서 얻어진 화합물 (13.47 g) 의 탈수 아세토니트릴 (350 ㎖) 현탁액에, 질소 분위기하, 분말상의 수산화칼륨 (10.3 g) 과 트리스[2-(2-메톡시에톡시)에틸]아민 (1.13 ㎖) 을 첨가하고, 실온에서 1.5 시간 교반하였다. 빙랭하, 이미 알려진 문헌 (J. Med. Chem. 1976, 19, 6, 814-816) 의 2,3,5-트리-O-벤질-α-D-아라비노퓨라노실클로라이드 (40.2 g) 의 아세토니트릴 (100 ㎖) 용액을 조금씩 첨가한 후, 실온까지 승온하여 4 시간 교반하였다. 불용물을 여과 제거하고, 아세토니트릴로 세정하였다. 여과액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 2 회 정제하고, 표제 화합물 (26.19 g) 을 얻었다. To a suspension of the compound (13.47 g) obtained in step 9 in dehydrated acetonitrile (350 ml), powdery potassium hydroxide (10.3 g) and tris[2-(2-methoxyethoxy)ethyl]amine ( 1.13 mL) was added and stirred at room temperature for 1.5 hours. Under ice cooling, 2,3,5-tri-O-benzyl-α-D-arabinofuranosylchloride (40.2 g) of known literature (J. Med. Chem. 1976, 19, 6, 814-816) After adding a solution of acetonitrile (100 ml) little by little, the temperature was raised to room temperature and stirred for 4 hours. Insoluble matter was filtered off and washed with acetonitrile. The filtrate was concentrated under reduced pressure, and the residue was purified twice by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (26.19 g).

Figure pat00509
Figure pat00509

(공정 11) (Process 11)

2-β-D-아라비노퓨라노실-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-β-D-arabinofuranosyl-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 10 에서 얻어진 화합물 (26.19 g) 의 탈수 디클로로메탄 (300 ㎖) 용액에, 질소 분위기하, -78 ℃ 에서 3염화붕소의 디클로로메탄 용액 (1M, 200 ㎖) 을 첨가하고, 동 온도에서 2 시간 교반한 후, 0 ℃ 까지 승온하여 추가로 4 시간 교반하였다. 반응액을 재차 -78 ℃ 까지 냉각시키고, 메탄올 (80 ㎖) 의 디클로로메탄 (160 ㎖) 용액을 첨가하고, 실온까지 승온하면서 30 분간 교반하였다. 반응액을 감압 농축하고, 에탄올로 2 회 공비하였다. 잔류물에 에탄올 (200 ㎖) 과 디에틸에테르 (100 ㎖) 를 첨가하여 슬러리상으로 하고, 고체를 여과 채취하였다 (고체 1). 여과액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축 후, 에탄올을 첨가하여 슬러리상으로 하고, 고체를 여과 채취하였다 (고체 2). 고체 1 과 고체 2 를 아울러 표제 화합물 (13.2 g) 을 얻었다.To a dehydrated dichloromethane (300 ml) solution of the compound (26.19 g) obtained in step 10 above, a dichloromethane solution of boron trichloride (1M, 200 ml) was added under a nitrogen atmosphere at -78°C, and After stirring for an hour, the temperature was raised to 0°C and further stirred for 4 hours. The reaction solution was cooled again to -78°C, a solution of methanol (80 ml) in dichloromethane (160 ml) was added, and the mixture was stirred for 30 minutes while heating to room temperature. The reaction solution was concentrated under reduced pressure and azeotroped twice with ethanol. Ethanol (200 ml) and diethyl ether (100 ml) were added to the residue to form a slurry, and the solid was collected by filtration (solid 1). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane/methanol]. After concentrating the fraction containing the target substance under reduced pressure, ethanol was added to form a slurry, and the solid was collected by filtration (solid 2). Solid 1 and Solid 2 were combined to obtain the title compound (13.2 g).

Figure pat00510
Figure pat00510

(공정 12) (Process 12)

2-[3,5-비스-O-(옥산-2-일)-β-D-아라비노퓨라노실]-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-[3,5-bis-O-(oxan-2-yl)-β-D-arabinofuranosyl]-2,7,8,9-tetrahydro-6-thia-2,3,5 -triazabenzo[cd]azulene

상기 공정 11 에서 얻어진 화합물 (15.35 g) 의 탈수 디메틸술폭시드 (160 ㎖) 용액에, 0 ℃ 에서 3,4-디하이드로-2H-피란 (17.2 ㎖) 과 p-톨루엔술폰산 1수화물 (9.02 g) 을 첨가하고, 실온에서 3 시간 교반하였다. 3,4-디하이드로-2H-피란 (8.6 ㎖) 을 추가하고, 45 분간 교반한 후, 곧바로 트리에틸아민 (13 ㎖) 을 첨가하여 반응을 정지시켰다. 반응액을 아세트산에틸과 포화 탄산수소나트륨 수용액의 2 층에 따르고, 아세트산에틸로 추출하였다. 유기층을 물로 1 회, 포화 식염수로 2 회 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (10.81 g) 을 4 종류의 디아스테레오머 혼합물로서 얻었다.To a solution of the compound (15.35 g) obtained in step 11 above in dehydrated dimethylsulfoxide (160 ml), 3,4-dihydro-2H-pyran (17.2 ml) and p-toluenesulfonic acid monohydrate (9.02 g) were added at 0°C. was added and stirred at room temperature for 3 hours. After adding 3,4-dihydro-2H-pyran (8.6 ml) and stirring for 45 minutes, triethylamine (13 ml) was added immediately to stop the reaction. The reaction solution was poured into two layers of ethyl acetate and saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was washed once with water and twice with saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (10.81 g) as a mixture of four diastereomers.

Figure pat00511
Figure pat00511

(공정 13) (Process 13)

2-[2-데옥시-2-플루오로-3,5-비스-O-(옥산-2-일)-β-D-리보퓨라노실]-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-[2-deoxy-2-fluoro-3,5-bis-O-(dioxane-2-yl)-β-D-ribofuranosyl]-2,7,8,9-tetrahydro- 6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 12 에서 얻어진 화합물 (10.81 g) 의 탈수 디클로로메탄 (150 ㎖) 용액에, 질소 분위기하, 0 ℃ 에서 피리딘 (5.3 ㎖) 과 트리플루오로메탄술폰산 무수물 (5.6 ㎖) 을 첨가하고, 동 온도에서 1 시간 교반하였다. 반응액에 빙편을 넣어 반응을 정지시킨 후, 반응액을 아세트산에틸과 포화 탄산수소나트륨 수용액의 2 층에 따르고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 2 회 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하고, 트리플레이트의 미정제체를 아모르퍼스로서 얻었다. 얻어진 트리플레이트의 미정제체를 탈수 테트라하이드로푸란 (150 ㎖) 에 용해하고, 빙랭하에서 불화테트라부틸암모늄의 테트라하이드로푸란 용액 (약 1 M, 154 ㎖) 을 조금씩 첨가하고, 동 온도에서 밤새 교반하였다. 반응액에 포화 염화암모늄 수용액을 첨가하여 반응을 정지시켰다. 반응액을 절반 정도의 양까지 감압 농축하였다. 잔류물을 아세트산에틸과 포화 염화암모늄 수용액의 2 층에 따르고, 아세트산에틸로 추출하였다. 유기층을 포화 염화암모늄 수용액으로 1 회, 포화 식염수로 2 회 세정하였다. 수층을 아세트산에틸로 재차 추출하고, 추출액을 포화 식염수로 세정하였다. 유기층을 아울러 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하고, 표제 화합물 (40.37 g) 의 미정제체를 얻었다. To a dehydrated dichloromethane (150 ml) solution of the compound (10.81 g) obtained in step 12 above, pyridine (5.3 ml) and trifluoromethanesulfonic anhydride (5.6 ml) were added under a nitrogen atmosphere at 0°C, and the mixture was heated at the same temperature. was stirred for 1 hour. After stopping the reaction by adding pieces of ice to the reaction solution, the reaction solution was poured into two layers of ethyl acetate and a saturated aqueous solution of sodium hydrogencarbonate, followed by extraction with ethyl acetate. The organic layer was washed twice with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude form of triflate as an amorphous substance. The obtained crude triflate was dissolved in dehydrated tetrahydrofuran (150 ml), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (about 1 M, 154 ml) was added little by little under ice-cooling, and the mixture was stirred overnight at the same temperature. A saturated aqueous solution of ammonium chloride was added to the reaction solution to stop the reaction. The reaction solution was concentrated under reduced pressure to about half the amount. The residue was poured into two layers of ethyl acetate and saturated aqueous ammonium chloride solution, and extracted with ethyl acetate. The organic layer was washed once with a saturated aqueous ammonium chloride solution and twice with saturated brine. The aqueous layer was extracted again with ethyl acetate, and the extract was washed with saturated brine. Together, the organic layers were dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product of the title compound (40.37 g).

MS(ESI)m/z : 494[M＋H]＋.MS(ESI)m/z : 494[M+H] + .

(공정 14) (Process 14)

2-(2-데옥시-2-플루오로-β-D-리보퓨라노실)-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo [cd] Azulene

상기 공정 13 에서 얻어진 화합물 (40.37 g) 의 메탄올 (400 ㎖) 용액에 p-톨루엔술폰산 1수화물 (2.09 g) 을 첨가하고, 60 ℃ 에서 4 시간 교반하였다. 반응액에 트리에틸아민 (16 ㎖) 을 첨가하여 반응을 정지시켰다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸 → 아세트산에틸/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 슬러리상이 될 때까지 감압 농축하고, 고체를 여과 채취하였다. 얻어진 고체를 헥산/아세트산에틸 (1 : 1) 로 세정하고, 고체 1 을 얻었다. 여과액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 고체 2 를 얻었다. 고체 1 과 고체 2 를 아울러 표제 화합물 (5.32 g) 을 얻었다.To a solution of the compound (40.37 g) obtained in step 13 in methanol (400 ml) was added p-toluenesulfonic acid monohydrate (2.09 g), and the mixture was stirred at 60°C for 4 hours. Triethylamine (16 ml) was added to the reaction solution to stop the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane/ethyl acetate→ethyl acetate/methanol]. A fraction containing the target substance was concentrated under reduced pressure until it became a slurry, and the solid was collected by filtration. The obtained solid was washed with hexane/ethyl acetate (1:1) to obtain a solid 1. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane/methanol] to obtain a solid 2. Solid 1 and Solid 2 were combined to obtain the title compound (5.32 g).

Figure pat00512
Figure pat00512

(공정 15) (Process 15)

2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-데옥시-2-플루오로-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-ribofuranosyl}-2,7,8,9- tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 14 에서 얻어진 화합물 (5.32 g) 을 사용하여, 실시예 11 공정 1 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물 (10.1 g) 을 얻었다.Using the compound (5.32 g) obtained in the above step 14, a reaction was carried out using the same method as in Example 11 step 1 to obtain the title compound (10.1 g).

Figure pat00513
Figure pat00513

(공정 16) (Process 16)

2-(5-O-[비스(4-메톡시페닐)(페닐)메틸]-3-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-2-데옥시-2-플루오로-β-D-리보퓨라노실)-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌 2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}- 2-deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

상기 공정 15 에서 얻어진 화합물 (10.1 g) 을 사용하여, 실시예 1 공정 6 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (12.6 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 1 : 1) 로서 얻었다.Using the compound (10.1 g) obtained in step 15 above, a reaction was carried out in the same manner as in step 6 of Example 1, and the title compound (12.6 g) was mixed with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 1:1). ) was obtained as

Figure pat00514
Figure pat00514

(공정 17) (Process 17)

상기 공정 16 에서 얻어진 화합물 (740 ㎎) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 22 공정 3 에서 얻어진 화합물 (924 ㎎) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (740 mg) obtained in step 16 above, reaction was carried out in the same manner as in step 7 of Example 1 to obtain 2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo aceto of )-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene A nitrile solution was obtained. Using the obtained acetonitrile solution and the compound (924 mg) obtained in Example 22 step 3, a reaction was carried out in the same manner as in Example 1 step 8, and the obtained crude product was used as it was for the next reaction.

(공정 18) (Process 18)

2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)- 14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-2-oxo-2-sulfanyl -10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 17 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (502 ㎎ : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 17 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (502 mg: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1065(M＋H)＋.MS(ESI) m/z : 1065(M+H) + .

(공정 19) (Process 19)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8, 9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-7-[1-(2-hydroxyethyl)- 6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[ 3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 18 에서 얻어진 화합물 (502 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법을 이용하여 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (67.8 ㎎ : 불순물 함유) 과 디아스테레오머 2 (69.5 ㎎ : 불순물 함유) 를 얻었다. Using the compound (502 mg) obtained in step 18 above, a reaction was carried out using the same method as in step 10 of Example 1 to obtain diastereomer 1 (67.8 mg: containing impurities) and diastereomer 2 (containing impurities) of the title compound. 69.5 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 908(M＋H)＋. MS(ESI) m/z : 908(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 908(M＋H)＋.MS(ESI) m/z : 908(M+H) + .

(공정 20-1) (Process 20-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2 (7H)-yl)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 19 에서 얻어진 화합물 (디아스테레오머 1) (67.8 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (67.8 mg: containing impurities) obtained in Step 19 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 25 ％ - 75 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile - methanol solution (1: 1), acetonitrile - methanol solution (1:1): 10% - 45% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 25% - 75% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (21.2 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (21.2 mg).

Figure pat00515
Figure pat00515

(공정 20-2) (Process 20-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2 (7H)-yl)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 19 에서 얻어진 화합물 (디아스테레오머 2) (69.5 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (69.5 mg: containing impurities) obtained in Step 19 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 25 ％ - 75 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 45% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 25% - 75% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (14.9 ㎎) 을 얻었다. The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (14.9 mg).

Figure pat00516
Figure pat00516

실시예 59 : CDN49 의 합성 Example 59: Synthesis of CDN49

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]- 14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10 -bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 210][Formula 210]

Figure pat00517
Figure pat00517

[합성 스킴][Synthesis Scheme]

[화학식 211][Formula 211]

Figure pat00518
Figure pat00518

(공정 1) (Process 1)

실시예 58 공정 16 에서 얻어진 화합물 (1.80 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 45 공정 3 에서 얻어진 화합물 (2.30 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.80 g) obtained in Example 58 Step 16, reaction was carried out in the same manner as in Example 1 Step 7, and 2-{2-deoxy-2-fluoro-3-O-[hydroxy (oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene of acetonitrile was obtained. Using the obtained acetonitrile solution and the compound (2.30 g) obtained in Example 45 Step 3, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-7-{1-[2-(1,3-디옥소-1,3-디하이드로-2H-이소인돌-2-일)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-일]옥시}프로판니트릴 3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-thia -2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-7-{1-[2-(1,3-dioxo-1,3-dihydro-2H-iso Indol-2-yl) ethyl] -6-oxo-1,6-dihydro-9H-purin-9-yl} -15-fluoro-2-oxo-2-sulfanyl-10-sulfanylidene octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -10-yl]oxy}propanenitrile

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.22 g) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 1 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (1.22 g) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1090(M＋H)＋.MS(ESI) m/z : 1090(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-16-{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6- Dihydro-9H-purin-9-yl]-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo [cd]azulen-2(7H)-yl)-15-fluoro-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 2 에서 얻어진 화합물 (1.22 g) 을 사용하여, 실시예 45 공정 6 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (108 ㎎ : 불순물 함유) 과 디아스테레오머 2 (111 ㎎ : 불순물 함유) 를 얻었다. Using the compound (1.22 g) obtained in step 2 above, the reaction was carried out in the same manner as in step 6 of Example 45, and then purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile]. Diastereomer 1 (108 mg: containing impurities) and diastereomer 2 (111 mg: containing impurities) of the title compound were obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 907(M＋H)＋. MS(ESI) m/z : 907(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 907(M＋H)＋.MS(ESI) m/z : 907(M+H) + .

(공정 4-1) (Step 4-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2 ,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 1) (108 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (108 mg: containing impurities) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 50 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 50% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (44.4 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (44.4 mg).

Figure pat00519
Figure pat00519

(공정 4-2) (Process 4-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-아미노에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl ]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2 ,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 2) (111 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (111 mg: containing impurities) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 20 ％ - 60 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 20% - 60% (0 min - 40 min)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (40.6 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (40.6 mg).

Figure pat00520
Figure pat00520

실시예 60 : CDN50 의 합성 Example 60: Synthesis of CDN50

N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)-2-하이드록시아세트아미드N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo [cd] Azulene-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5 ,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6- oxo-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[화학식 212][Formula 212]

Figure pat00521
Figure pat00521

[합성 스킴][Synthesis Scheme]

[화학식 213][Formula 213]

Figure pat00522
Figure pat00522

(공정 1-1) (Process 1-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2 (7H)-yl)-15-fluoro-16-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6-oxo-1,6-dihydro-9H-purine -9-yl} -2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

실시예 59 공정 4-1 에서 얻어진 화합물 (20.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (20.0 mg) obtained in Example 59 Step 4-1, the reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification Conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 30 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 10% - 30% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (15.6 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (15.6 mg).

Figure pat00523
Figure pat00523

(공정 1-2) (Process 1-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-7-{1-[2-(2-하이드록시아세트아미드)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2 (7H)-yl)-15-fluoro-16-hydroxy-7-{1-[2-(2-hydroxyacetamide)ethyl]-6-oxo-1,6-dihydro-9H-purine -9-yl} -2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

실시예 59 공정 4-2 에서 얻어진 화합물 (10.0 ㎎) 을 사용하여, 실시예 7 공정 1-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (10.0 mg) obtained in Example 59 Step 4-2, reaction was carried out in the same manner as in Example 7 Step 1-1, and then purified under the following [Purification Conditions] to obtain the title compound with triethylamine obtained as a salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 7 ％ - 25 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 7% - 25% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (6.6 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (6.6 mg).

Figure pat00524
Figure pat00524

실시예 61 : CDN51 의 합성 Example 61: Synthesis of CDN51

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-비스(술파닐)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-14-(8 ,9-dihydro-6-thia-2,3,5-triazabenzo [cd] azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2,10-bis (alcohol panyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-dione

[화학식 214][Formula 214]

Figure pat00525
Figure pat00525

[합성 스킴][Synthesis Scheme]

[화학식 215][Formula 215]

Figure pat00526
Figure pat00526

(공정 1) (Process 1)

실시예 58 공정 16 에서 얻어진 화합물 (1.80 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-2,7,8,9-테트라하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 47 공정 6 에서 얻어진 화합물 (3.10 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.80 g) obtained in Example 58 Step 16, reaction was carried out in the same manner as in Example 1 Step 7, and 2-{2-deoxy-2-fluoro-3-O-[hydroxy (oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene of acetonitrile was obtained. Using the obtained acetonitrile solution and the compound (3.10 g) obtained in Example 47 Step 6, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-(트리메틸실릴)에틸[2-({6-벤즈아미드-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]카르바메이트 2-(trimethylsilyl)ethyl[2-({6-benzamide-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl] Oxy}-10-(2-cyanoethoxy)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl) -15-fluoro-2-oxo-2-sulfanyl-10-sulfanylidene octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]carbamate

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.83 g : 불순물 함유) 을 얻었다.Using the crude product obtained in step 1 above, reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (1.83 g: containing impurities).

MS(ESI)m/z : 1222(M＋H)＋.MS(ESI) m/z : 1222(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-아미노-2-{[2-({[2-(트리메틸실릴)에톡시]카르보닐}아미노)에틸]아미노}-9H-푸린-9-일)-16-{[tert-부틸(디메틸)실릴]옥시}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-2-{[2-({[2-(trimethyl) silyl)ethoxy]carbonyl}amino)ethyl]amino}-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6 -Thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-2,10-dioxooctahydro-2H,10H,12H-5,8- Methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 2 에서 얻어진 혼합물 (1.83 g) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (151 ㎎ : 불순물 함유) 과 디아스테레오머 2 (103 ㎎ : 불순물 함유) 를 얻었다. Using the mixture (1.83 g) obtained in step 2 above, a reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (151 mg: containing impurities) and diastereomer 2 (103 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1065(M＋H)＋. MS(ESI) m/z : 1065(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1065(M＋H)＋.MS(ESI) m/z : 1065(M+H) + .

(공정 4-1) (Step 4-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-14- (8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-di oxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 1) (Diastereomer 1)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 1) (151 ㎎ : 불순물 함유) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (151 mg: containing impurity) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 40 Step 5, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (10.6 ㎎) 을 얻었다.The resulting triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (10.6 mg).

Figure pat00527
Figure pat00527

(공정 4-2) (Process 4-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-아미노-2-[(2-아미노에틸)아미노]-9H-푸린-9-일}-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-14- (8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-di oxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecine-2,10-bis(thiolate)

(디아스테레오머 2) (Diastereomer 2)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 2) (103 ㎎ : 불순물 함유) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 2) (103 mg: containing impurities) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 40 Step 5, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 30 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 10% - 30% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (12.1 ㎎) 을 얻었다.The resulting triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (12.1 mg).

Figure pat00528
Figure pat00528

실시예 62 : CDN52 의 합성 Example 62: Synthesis of CDN52

(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-2,16-디하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-10-술파닐-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1, 6-dihydro-9H-purin-9-yl]-10-sulfanyl-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 216][Formula 216]

Figure pat00529
Figure pat00529

[합성 스킴][Synthesis Scheme]

[화학식 217][Formula 217]

Figure pat00530
Figure pat00530

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 22 공정 3 에서 얻어진 화합물 (1.13 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.00 g) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (1.13 g) obtained in Example 22 Step 3, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-하이드록시-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-hydride Roxy-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9, 11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 1 에서 얻어진 미정제 생성물의 피리딘 (32.5 ㎖) 용액을 25 ㎖ 정도까지 농축 후, 2-클로로-5,5-디메틸-1,3,2λ5-디옥사포스피난-2-온 (945 ㎎) 을 첨가하고, 실온에서 30 분간 교반하였다. 반응액에 요오드 (1.11 g) 를 첨가하고, 1 시간 교반하였다. 반응액을 탄산수소나트륨 (4.30 g) 의 수용액 (150 ㎖) 에 따르고, 30 분간 교반 후, 아세트산에틸로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올] 로 정제하고, 표제 화합물 (671 ㎎ : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. After concentrating the pyridine (32.5 ml) solution of the crude product obtained in step 1 to about 25 ml, 2-chloro-5,5-dimethyl-1,3,2λ 5 -dioxaphosphinan-2-one (945 mg) was added and stirred at room temperature for 30 minutes. Iodine (1.11 g) was added to the reaction solution, and the mixture was stirred for 1 hour. The reaction solution was poured into an aqueous solution (150 ml) of sodium hydrogen carbonate (4.30 g), and after stirring for 30 minutes, extraction was performed with ethyl acetate. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol] to give the title compound (671 mg: containing impurities) as a diastereomer mixture on a phosphorus atom.

MS(ESI)m/z : 1136(M＋H)＋.MS(ESI)m/z: 1136(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-10-sulfide-14-(6, 7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2-oleate

상기 공정 2 에서 얻어진 화합물 (671 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (55.6 ㎎ : 불순물 함유) 과 디아스테레오머 2 (65.7 ㎎ : 불순물 함유) 를 얻었다. Using the compound (671 mg) obtained in step 2 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (55.6 mg: containing impurities) and diastereomer 2 (65.7 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 875(M＋H)＋. MS(ESI) m/z : 875(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 875(M＋H)＋.MS(ESI) m/z : 875(M+H) + .

(공정 4-1) (Step 4-1)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl]-2,10-dioxo-10-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2-oleate

(디아스테레오머 1) (Diastereomer 1)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 1) (55.6 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (Diastereomer 1) (55.6 mg: containing impurities) obtained in Step 3 above, the reaction was carried out in the same manner as in Example 1 Step 11, and then purified under the following [Purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 5 ％ - 25 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 5% - 25% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (11.3 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (11.3 mg).

Figure pat00531
Figure pat00531

(공정 4-2) (Process 4-2)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl]-2,10-dioxo-10-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-2-oleate

(디아스테레오머 2) (Diastereomer 2)

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 2) (65.7 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (diastereomer 2) (65.7 mg: containing impurities) obtained in step 3 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound Obtained as triethylamine salt.

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 3 ％ - 20 ％ (0 분 - 30 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 3% - 20% (0 min - 30 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (23.5 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (23.5 mg).

Figure pat00532
Figure pat00532

실시예 63 : CDN53 의 합성 Example 63: Synthesis of CDN53

(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-10,16-디하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2-술파닐-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-10,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1, 6-dihydro-9H-purin-9-yl]-2-sulfanyl-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecine-2,10-dione

[화학식 218][Formula 218]

Figure pat00533
Figure pat00533

[합성 스킴][Synthesis Scheme]

[화학식 219][Formula 219]

Figure pat00534
Figure pat00534

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (1.02 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다 (아세토니트릴 용액 A). 실시예 22 공정 3 에서 얻어진 화합물 (1.23 g) 을 탈수 아세토니트릴 (10 ㎖) 로 3 회 공비 탈수하였다. 마지막 1 회는 7 ㎖ 정도의 아세토니트릴을 남기고, 몰레큘러 시브스 3A, 1/16 (펠릿상의 5 립) 을 첨가하였다 (아세토니트릴 용액 B). 아세토니트릴 용액 A 와 아세토니트릴 용액 B 를 혼합하고, 질소 분위기하, 실온에서 15 분간 교반하였다. 반응액에 tert-부틸하이드로퍼옥사이드의 데칸 용액 (5.5 M, 0.50 ㎖) 을 첨가하고, 40 분간 교반 후, 반응액을 빙랭하고, 티오황산나트륨 5 수화물 (826 ㎎) 의 수용액 (1.1 ㎖) 을 첨가하고, 10 분간 교반하였다. 반응액에 물 (20 ㎖) 을 첨가하고, 디클로로메탄-메탄올 혼합액으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물의 디클로로메탄 (15.9 ㎖) 용액에 물 (0.200 ㎖) 과 디클로로아세트산 (1.00 ㎖) 의 디클로로메탄 (15.9 ㎖) 용액을 차례로 첨가하고, 실온에서 15 분간 교반하였다. 반응액에 피리딘 (11.0 ㎖) 을 첨가하여 반응을 정지 후, 반응액을 감압 농축하였다. 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.02 g) obtained in Example 44 Step 8, a reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained (acetonitrile solution A). The compound (1.23 g) obtained in Example 22 step 3 was subjected to azeotropic dehydration 3 times with dehydrated acetonitrile (10 ml). For the last one, Molecular Sieves 3A, 1/16 (5 granules in the form of pellets) were added leaving about 7 ml of acetonitrile (acetonitrile solution B). Acetonitrile solution A and acetonitrile solution B were mixed and stirred for 15 minutes at room temperature under a nitrogen atmosphere. A decane solution of tert-butyl hydroperoxide (5.5 M, 0.50 ml) was added to the reaction solution, and after stirring for 40 minutes, the reaction solution was ice-cooled, and an aqueous solution of sodium thiosulfate pentahydrate (826 mg) (1.1 ml) was added. and stirred for 10 minutes. Water (20 ml) was added to the reaction mixture, and extraction was performed with a mixture of dichloromethane and methanol. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. To a dichloromethane (15.9 ml) solution of the residue was added water (0.200 ml) and a dichloromethane (15.9 ml) solution of dichloroacetic acid (1.00 ml) in that order, and the mixture was stirred at room temperature for 15 minutes. After stopping the reaction by adding pyridine (11.0 ml) to the reaction solution, the reaction solution was concentrated under reduced pressure. The obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

N,N-디에틸에탄아미늄 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{1-[2-(벤조일옥시)에틸]-6-옥소-1,6-디하이드로-9H-푸린-9-일}-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-티올레이트 N,N-diethylethanelaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{1-[2-(benzoyloxy)ethyl]-6-oxo-1,6- Dihydro-9H-purin-9-yl} -14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2 -yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2,10-dioxoooctahydro-2H,10H,12H- 5,8-methano- 2λ 5,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate

상기 공정 1 에서 얻어진 미정제 생성물을, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (122 ㎎ : 불순물 함유) 을 얻었다. The crude product obtained in step 1 was reacted in the same manner as in step 9 of Example 1 to obtain the title compound (122 mg: containing impurities).

MS(ESI)m/z : 1136(M＋H)＋.MS(ESI)m/z: 1136(M+H) + .

(공정 3) (Process 3)

(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-10-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2-술파닐-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-10-hydroxy-7-[1-(2 -Hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2-sulfanyl-14-(6,7,8,9-tetrahydro-2H-2,3 ,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1 ,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

상기 공정 2 에서 얻어진 화합물 (122 mg, 불순물 함유) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다. Using the compound (122 mg, containing impurities) obtained in step 2 above, a reaction was carried out in the same manner as in step 10 of Example 1, and the obtained crude product was used as it was in the next reaction.

MS(ESI)m/z : 875(M＋H)＋.MS(ESI) m/z : 875(M+H) + .

(공정 4) (Process 4)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-올레이트 Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl]-2,10-dioxo-2-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetra Azabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9 ,11,2,10]pentaoxadiphosphacyclotetradecyn-10-oleate

상기 공정 3 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 2 ％ - 30 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. 얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (30 ㎎) 을 얻었다. Using the crude product obtained in step 3 above, reaction was carried out in the same manner as in step 11 of Example 1, followed by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 2% - 30% (0 min - 30 min)] to obtain the title compound as a triethylamine salt. The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (30 mg).

Figure pat00535
Figure pat00535

실시예 64 : CDN54 의 합성 Example 64: Synthesis of CDN54

(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-2,10,16-트리하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-디온(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-7-[1-(2-hydroxyethyl)-6-oxo- 1,6-dihydro-9H-purin-9-yl] -14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulene-2 -yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxa Diphosphacyclotetradecine-2,10-dione

[화학식 220][Formula 220]

Figure pat00536
Figure pat00536

[합성 스킴][Synthesis Scheme]

[화학식 221][Formula 221]

Figure pat00537
Figure pat00537

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (1.00 g) 과 실시예 22 공정 3 에서 얻어진 화합물 (1.13 g) 을 사용하여, 실시예 63 공정 1 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.00 g) obtained in Step 8 of Example 44 and the compound (1.13 g) obtained in Step 3 of Example 22, a reaction was carried out in the same manner as in Example 63 Step 1, and the obtained crude product was obtained as follows. was used for the reaction.

(공정 2) (Process 2)

2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸 벤조에이트 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5; 6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-hydride hydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2 ,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 62 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (602 ㎎ : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 1 above, a reaction was carried out in the same manner as in step 2 of Example 62 to obtain the title compound (602 mg: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1120(M＋H)＋.MS(ESI) m/z : 1120(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(oleate)

상기 공정 2 에서 얻어진 화합물 (602 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (90.8 ㎎ : 불순물 함유) 을 얻었다. Using the compound (602 mg) obtained in step 2 above, reaction was carried out in the same manner as in step 10 of Example 1 to obtain the title compound (90.8 mg: containing impurities).

MS(ESI)m/z : 859(M＋H)＋.MS(ESI) m/z : 859(M+H) + .

(공정 4) (Process 4)

디나트륨 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-7-[1-(2-하이드록시에틸)-6-옥소-1,6-디하이드로-9H-푸린-9-일]-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(올레이트) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6 -dihydro-9H-purin-9-yl] -2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-2,10-bis(oleate)

상기 공정 3 에서 얻어진 화합물 (90.8 ㎎ : 불순물 함유) 을 사용하여, 실시예 1 공정 11 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물을 트리에틸아민염으로서 얻었다. Using the compound (90.8 mg: containing impurities) obtained in step 3 above, the reaction was carried out in the same manner as in step 11 of Example 1, and then purified under the following [purification conditions] to obtain the title compound as a triethylamine salt. .

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 1 ％ - 20 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 1% - 20% (0 min - 40 minute)].

얻어진 트리에틸아민염을, 실시예 1 공정 11 에 기재된 [나트륨염으로의 변환] 과 동일한 수법으로 염 교환을 실시하고, 표제 화합물 (40.4 ㎎) 을 얻었다.The obtained triethylamine salt was salt-exchanged in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (40.4 mg).

Figure pat00538
Figure pat00538

실시예 65 : 약물 링커 5 의 합성 Example 65: Synthesis of Drug Linker 5

[합성 스킴][Synthesis Scheme]

[화학식 222][Formula 222]

Figure pat00539
Figure pat00539

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-비스{[tert-부틸(디메틸)실릴]옥시}-7-(6-{[(글리실아미노)메톡시]메틸}-9H-푸린-9-일)-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7- (6-{[(glycylamino)methoxy]methyl}-9H-purin-9-yl)-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2; 3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][ 1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

실시예 17 공정 7 에서 얻어진 화합물 (디아스테레오머 2) (80.6 ㎎) 을 사용하여, 실시예 22 공정 7-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (66.1 ㎎ : 불순물 함유) 을 얻었다. Using the compound (diastereomer 2) (80.6 mg) obtained in Example 17 Step 7, a reaction was carried out in the same manner as in Example 22 Step 7-1 to obtain the title compound (66.1 mg: containing impurities).

MS(ESI)m/z : 1059(M＋H)＋.MS(ESI)m/z: 1059(M+H) + .

(공정 2) (Process 2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-{[(글리실아미노)메톡시]메틸}-9H-푸린-9-일)-15,16-디하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-{[(glycylamino)methoxy]methyl}-9H- Purin-9-yl) -15,16-dihydroxy-2,10-dioxo-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [ cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 1 에서 얻어진 화합물 (66.1 ㎎) 을 사용하여, 실시예 22 공정 8-1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (40.4 ㎎ : 불순물 함유) 을 얻었다. Using the compound (66.1 mg) obtained in the above step 1, a reaction was carried out in the same manner as in Example 22 step 8-1 to obtain the title compound (40.4 mg: containing impurities).

MS(ESI)m/z : 831(M＋H)＋.MS(ESI) m/z : 831(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[({9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-6-일}메톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[({9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo- 2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H ,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl ]-9H-purin-6-yl}methoxy)methyl]glycinamide

(약물 링커 5) (drug linker 5)

상기 공정 2 에서 얻어진 화합물 (40.4 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 45 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (5.0 ㎎) 을 얻었다. Using the compound (40.4 mg) obtained in step 2 above, reaction was carried out in the same manner as in step 9-1 of Example 22, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] and fractionation Purification was performed by HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% - 45% (0 min - 30 min)] to obtain the title compound (5.0 mg).

Figure pat00540
Figure pat00540

실시예 66 : 약물 링커 6 의 합성 Example 66: Synthesis of Drug Linker 6

[합성 스킴][Synthesis Scheme]

[화학식 223][Formula 223]

Figure pat00541
Figure pat00541

(공정 1) (Process 1)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-({2-[(2,5-디옥소피롤리딘-1-일)옥시]-2-옥소에톡시}메틸)글리신아미드 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-( {2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethoxy}methyl)glycinamide

이미 알려진 문헌 (WO2014/057687) 의 {[(N-{[(9H-플루오렌-9-일)메톡시]카르보닐}글리실)아미노]메톡시}아세트산 (955 ㎎) 의 N,N-디메틸포름아미드 (8.0 ㎖) 용액에, 1,8-디아자비시클로[5.4.0]-7-운데센 (0.74 ㎖) 을 첨가하고, 실온에서 1 시간 교반하였다 (반응액 A). 실시예 22 공정 10 에서 얻어진 화합물 (938 ㎎) 의 N,N-디메틸포름아미드 (8.0 ㎖) 용액에 N-하이드록시숙신이미드 (229 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (380 ㎎) 을 첨가하고, 실온에서 50 분간 교반하였다 (반응액 B). 반응액 A 를 반응액 B 에 첨가하고, 실온에서 1 시간 교반하였다. 반응액에 디클로로메탄 (50 ㎖) 과 10 ％ 시트르산 수용액 (10 ㎖) 을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/(클로로포름/메탄올/물 = 7 : 3 : 1 의 하층)] 으로 정제하였다. 얻어진 화합물의 N,N-디메틸포름아미드 (8.0 ㎖) 용액에, N-하이드록시숙신이미드 (229 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (380 ㎎) 을 첨가하고, 실온에서 30 분간 교반하였다. 반응액에 디클로로메탄 (100 ㎖) 과 물 (25 ㎖) 을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축하고, 잔류물에 디에틸에테르를 첨가하여 슬러리상으로 하였다. 얻어진 고체를 여과 채취하고, 표제 화합물 (412 ㎎) 을 얻었다.N,N- of {[(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycyl)amino]methoxy}acetic acid (955 mg) of known literature (WO2014/057687) To a solution of dimethylformamide (8.0 ml), 1,8-diazabicyclo[5.4.0]-7-undecene (0.74 ml) was added, and the mixture was stirred at room temperature for 1 hour (reaction solution A). Example 22 N-hydroxysuccinimide (229 mg) and 1-ethyl-3-(3-dimethylaminopropyl) were added to a solution of the compound (938 mg) obtained in step 10 in N,N-dimethylformamide (8.0 ml). )-carbodiimide hydrochloride (380 mg) was added, and the mixture was stirred at room temperature for 50 minutes (reaction solution B). Reaction solution A was added to reaction solution B, and it stirred at room temperature for 1 hour. Dichloromethane (50 ml) and 10% citric acid aqueous solution (10 ml) were added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [lower layer of chloroform/(chloroform/methanol/water = 7:3:1)]. N, N-dimethylformamide (8.0 ml) solution of the obtained compound, N-hydroxysuccinimide (229 mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (380 mg) ) was added and stirred at room temperature for 30 minutes. Dichloromethane (100 ml) and water (25 ml) were added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [chloroform/methanol]. A fraction containing the target substance was concentrated under reduced pressure, and diethyl ether was added to the residue to form a slurry. The obtained solid was collected by filtration to obtain the title compound (412 mg).

Figure pat00542
Figure pat00542

(공정 2) (Process 2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)아미노]-2-옥소에톡시}메틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2 ,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)amino]-2-oxoethoxy}methyl)glycinamide

(약물 링커 6) (Drug Linker 6)

실시예 5 공정 8-2 에서 얻어진 화합물 (20.0 ㎎) 과 상기 공정 1 에서 얻어진 화합물 (23.7 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 30 ％ - 80 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (14.1 ㎎) 을 얻었다. Example 5 Using the compound (20.0 mg) obtained in Step 8-2 and the compound (23.7 mg) obtained in Step 1 above, a reaction was carried out in the same manner as in Example 21 Step 4, followed by C18 silica gel column chromatography [ 10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% - 45% (0 min - 40 min)], and preparative HPLC [10 mM acetic acid Triethylammonium aqueous solution/methanol, methanol: 30% - 80% (0 min - 40 min)] to obtain the title compound (14.1 mg).

Figure pat00543
Figure pat00543

실시예 67 : 약물 링커 7 의 합성 Example 67: Synthesis of Drug Linker 7

[합성 스킴][Synthesis Scheme]

[화학식 224][Formula 224]

Figure pat00544
Figure pat00544

(공정 1) (Process 1)

[(N-{[(9H-플루오렌-9-일)메톡시]카르보닐}-L-이소류실)아미노]메틸 아세테이트 [(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-isoleucyl)amino]methyl acetate

시판 (IRIS) 되는 N-{[(9H-플루오렌-9-일)메톡시]카르보닐}-L-이소류실글리신 (2.50 g) 의 테트라하이드로푸란 (45 ㎖) - 톨루엔 (15 ㎖) 혼합액에, 실온에서 피리딘 (0.588 ㎖) 과 4아세트산납 (3.24 g) 을 첨가하고, 65 ℃ 에서 3 시간 교반하였다. 반응액을 아세트산에틸로 희석하고, 물과 포화 식염수로 차례로 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (2.06 g) 을 얻었다. Commercially available (IRIS) mixture of N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-isoleucylglycine (2.50 g) in tetrahydrofuran (45 ml) - toluene (15 ml) Then, pyridine (0.588 ml) and lead tetraacetate (3.24 g) were added at room temperature, and the mixture was stirred at 65°C for 3 hours. The reaction solution was diluted with ethyl acetate and washed with water and saturated brine in that order. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (2.06 g).

Figure pat00545
Figure pat00545

(공정 2) (Process 2)

벤질 {[(N-{[(9H-플루오렌-9-일)메톡시]카르보닐}-L-이소류실)아미노]메톡시}아세테이트 Benzyl {[(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-isoleucyl)amino]methoxy}acetate

상기 공정 1 에서 얻어진 화합물 (2.06 g) 의 테트라하이드로푸란 (48 ㎖) 현탁액에, 0 ℃ 에서 글리콜산벤질 (1.38 ㎖) 과 p-톨루엔술폰산 1수화물 (92.3 ㎎) 을 첨가하고, 실온에서 4 시간 교반하였다. 반응액을 아세트산에틸로 희석하고, 포화 탄산수소나트륨수로 세정 후, 수층을 아세트산에틸로 추출하였다. 유기층을 아울러, 포화 식염수로 세정하고, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (1.72 g) 을 얻었다.To a suspension of the compound (2.06 g) obtained in step 1 above in tetrahydrofuran (48 ml), benzyl glycolate (1.38 ml) and p-toluenesulfonic acid monohydrate (92.3 mg) were added at 0°C, followed by incubation at room temperature for 4 hours. Stir. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium hydrogencarbonate, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (1.72 g).

Figure pat00546
Figure pat00546

(공정 3) (Process 3)

N-[(벤질옥시)카르보닐]글리실글리실-L-프롤릴-N-{[2-(벤질옥시)-2-옥소에톡시]메틸}-L-이소류신아미드 N-[(benzyloxy)carbonyl]glycylglycyl-L-prolyl-N-{[2-(benzyloxy)-2-oxoethoxy]methyl}-L-isoleucinamide

상기 공정 2 에서 얻어진 화합물 (1.72 g) 의 아세토니트릴 (40 ㎖) 현탁액에, 실온에서 1,8-디아자비시클로[5.4.0]-7-운데센 (0.290 ㎖) 을 첨가하고, 1 시간 교반하였다 (반응액 A). 시판되는 N-[(벤질옥시)카르보닐]글리실글리실-L-프롤린 (1.41 g) 의 아세토니트릴 (20 ㎖) 현탁액에, 실온에서 3H-[1,2,3]트리아졸[4,5-b]피리딘-3-올 (529 ㎎), 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (746 ㎎), 및 N,N-디이소프로필에틸아민 (0.678 ㎖) 을 첨가하고, 1 시간 교반하였다. 이 반응액을 실온에서 상기 반응액 A 에 첨가하고, 6 시간 교반 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하고, 표제 화합물 (1.56 g) 을 얻었다.To a suspension of the compound (1.72 g) obtained in step 2 above in acetonitrile (40 ml) was added 1,8-diazabicyclo[5.4.0]-7-undecene (0.290 ml) at room temperature, followed by stirring for 1 hour. (reaction solution A). To a commercially available suspension of N-[(benzyloxy)carbonyl]glycylglycyl-L-proline (1.41 g) in acetonitrile (20 ml), 3H-[1,2,3]triazole[4, 5-b]pyridin-3-ol (529 mg), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (746 mg), and N,N-diisopropylethylamine (0.678 mL ) was added and stirred for 1 hour. This reaction liquid was added to the reaction liquid A at room temperature, and after stirring for 6 hours, it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [chloroform/methanol] to give the title compound (1.56 g).

Figure pat00547
Figure pat00547

(공정 4) (Process 4)

글리실글리실-L-프롤릴-N-[(카르복시메톡시)메틸]-L-이소류신아미드 Glycylglycyl-L-prolyl-N-[(carboxymethoxy)methyl]-L-isoleucinamide

상기 공정 3 에서 얻어진 화합물 (1.56 g) 의 메탄올 (8 ㎖) - 테트라하이드로푸란 (24 ㎖) - 디클로로메탄 (8 ㎖) 혼합액에, 10 ％ 팔라듐탄소 (M) wet (1.4 g) 를 첨가하고, 수소 분위기하, 실온에서 23 시간 교반하였다. 반응액에 메탄올/테트라하이드로푸란 (1 : 1) 혼합액 (50 ㎖) 을 첨가하고, 셀라이트로 여과하였다. 셀라이트를 메탄올/테트라하이드로푸란 (1 : 1) 혼합액으로 세정하였다. 여과액을 감압 농축 후, 잔류물에 메탄올 (20 ㎖), 테트라하이드로푸란 (20 ㎖), 및 10 ％ 팔라듐탄소 (M) wet (1.0 g) 를 첨가하고, 수소 분위기하, 실온에서 3 시간 교반하였다. 반응액을 셀라이트로 여과 후, 셀라이트를 메탄올/테트라하이드로푸란 (1 : 1) 으로 세정하였다. 여과액을 감압 농축하고, 표제 화합물의 미정제체 (1.02 g) 를 얻었다.To a mixture of methanol (8 ml) - tetrahydrofuran (24 ml) - dichloromethane (8 ml) of the compound (1.56 g) obtained in step 3 above, 10% palladium carbon (M) wet (1.4 g) was added, The mixture was stirred for 23 hours at room temperature under a hydrogen atmosphere. A mixture of methanol/tetrahydrofuran (1:1) (50 ml) was added to the reaction mixture, and the mixture was filtered through celite. Celite was washed with a mixture of methanol/tetrahydrofuran (1:1). After concentrating the filtrate under reduced pressure, methanol (20 ml), tetrahydrofuran (20 ml), and 10% palladium carbon (M) wet (1.0 g) were added to the residue, and the mixture was stirred for 3 hours at room temperature under a hydrogen atmosphere. did After filtering the reaction solution through celite, the celite was washed with methanol/tetrahydrofuran (1:1). The filtrate was concentrated under reduced pressure to obtain a crude product of the title compound (1.02 g).

MS(ESI)m/z : 430(M＋H)＋.MS(ESI) m/z : 430(M+H) + .

(공정 5) (Process 5)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-[(카르복시메톡시)메틸]-L-이소류신아미드 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-prolyl-N-[( carboxymethoxy)methyl]-L-isoleucinamide

상기 공정 4 에서 얻어진 화합물 (1.02 g) 의 N,N-디메틸포름아미드 (24 ㎖) 용액에, 1-{[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]옥시}피롤리딘-2,5-디온 (956 ㎎) 과 N,N-디이소프로필에틸아민 (0.496 ㎖) 을 첨가하고, 실온에서 1 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축 후, 잔류물에 아세트산에틸을 첨가하여 고화시켰다. 얻어진 고체를 여과 채취하고, 표제 화합물 (1.06 g) 을 얻었다.1-{[4-(11,12-didehydrodibenzo[b,f]azocine-5( 6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (956 mg) and N,N-diisopropylethylamine (0.496 ml) were added and stirred at room temperature for 1 hour did The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [chloroform/methanol]. After concentration under reduced pressure, the fraction containing the target substance was solidified by adding ethyl acetate to the residue. The obtained solid was collected by filtration to obtain the title compound (1.06 g).

Figure pat00548
Figure pat00548

(공정 6) (Process 6)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-({2-[(2,5-디옥소피롤리딘-1-일)옥시]-2-옥소에톡시}메틸)-L-이소류신아미드 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-prolyl-N-({ 2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethoxy}methyl)-L-isoleucinamide

상기 공정 5 에서 얻어진 화합물 (1.06 g) 의 N,N-디메틸포름아미드 (16 ㎖) 현탁액에, N-하이드록시숙신이미드 (187 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (312 ㎎) 을 첨가하고, 실온에서 16 시간 교반하였다. 반응액을 클로로포름으로 희석하고, 물로 3 회 세정하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물에 아세트산에틸을 첨가하고, 얻어진 고체를 여과 채취하였다. 여과 채취한 고체에 디에틸에테르를 첨가하고, 슬러리상으로 한 후, 고체를 여과 채취하여, 표제 화합물 (767 ㎎) 을 얻었다.To a suspension of the compound (1.06 g) obtained in step 5 above in N,N-dimethylformamide (16 ml), N-hydroxysuccinimide (187 mg) and 1-ethyl-3-(3-dimethylaminopropyl) - Carbodiimide hydrochloride (312 mg) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with chloroform and washed three times with water. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the residue, and the obtained solid was collected by filtration. Diethyl ether was added to the solid collected by filtration to form a slurry, and then the solid was collected by filtration to obtain the title compound (767 mg).

Figure pat00549
Figure pat00549

(공정 7) (Process 7)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)아미노]-2-옥소에톡시}메틸)-L-이소류신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycyl-L-prolyl-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2, 10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyn-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)amino]-2-oxoethoxy}methyl)-L-isoleucinamide

(약물 링커 7) (Drug Linker 7)

실시예 5 공정 8-2 에서 얻어진 화합물 (20.0 ㎎) 과 상기 공정 6 에서 얻어진 화합물 (20.9 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 30 ％ - 80 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (11.9 ㎎) 을 얻었다.Example 5 Using the compound (20.0 mg) obtained in Step 8-2 and the compound (20.9 mg) obtained in Step 6 above, a reaction was carried out in the same manner as in Example 21 Step 4, followed by C18 silica gel column chromatography [ 10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% - 45% (0 min - 40 min)], and preparative HPLC [10 mM acetic acid Triethylammonium aqueous solution/methanol, methanol: 30% - 80% (0 min - 40 min)] to obtain the title compound (11.9 mg).

Figure pat00550
Figure pat00550

실시예 68 : 약물 링커 8 의 합성 Example 68: Synthesis of Drug Linker 8

[합성 스킴][Synthesis Scheme]

[화학식 225][Formula 225]

Figure pat00551
Figure pat00551

(공정 1) (Process 1)

N-(tert-부톡시카르보닐)글리실글리실-L-발릴-L-알라닌 N-(tert-butoxycarbonyl)glycylglycyl-L-valyl-L-alanine

시판 (ChemFun) 되는 N-(tert-부톡시카르보닐)글리실글리신 (5.00 g) 과 N-하이드록시숙신이미드 (2.97 g) 의 N,N-디메틸포르미아미드 (50 ㎖) 용액에, 질소 분위기하, 0 ℃ 에서 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (4.95 g) 을 첨가하고, 실온으로 승온하여 1 시간 교반하였다. 반응액에 트리에틸아민 (3.6 ㎖) 과 시판 (고쿠산 화학) 되는 L-발릴-L-알라닌 (4.05 g) 을 첨가하고, 동 온도 6 시간 교반하였다. 반응액을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하였다. 목적물을 포함하는 프랙션을 감압 농축하고, 디에틸에테르를 첨가하여 슬러리상으로 하였다. 얻어진 고체를 여과 채취하고, 표제 화합물 (4.99 g) 을 얻었다.In a solution of commercially available (ChemFun) N-(tert-butoxycarbonyl)glycylglycine (5.00 g) and N-hydroxysuccinimide (2.97 g) in N,N-dimethylformiamide (50 ml), Under a nitrogen atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (4.95 g) was added at 0°C, the temperature was raised to room temperature, and the mixture was stirred for 1 hour. Triethylamine (3.6 ml) and commercially available (Kokusan Chemical) L-valyl-L-alanine (4.05 g) were added to the reaction solution, and the mixture was stirred at the same temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane/methanol]. A fraction containing the target substance was concentrated under reduced pressure, and diethyl ether was added to form a slurry. The obtained solid was collected by filtration to obtain the title compound (4.99 g).

Figure pat00552
Figure pat00552

(공정 2) (Process 2)

N-(아자니우밀아세틸)글리실-L-발릴-L-알라닌 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-valyl-L-alanine trifluoroacetate

상기 공정 1 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.10 g) 의 미정제체를 얻었다.Using the compound (1.00 g) obtained in the above step 1, reaction was carried out in the same manner as in Example 21 step 1 to obtain a crude product of the title compound (1.10 g).

Figure pat00553
Figure pat00553

(공정 3) (Process 3)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-L-알라닌 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-L-alanine

상기 공정 2 에서 얻어진 화합물 (861 ㎎) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (737 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 2 using the compound (861 mg) obtained in Step 2 above to obtain the title compound (737 mg).

Figure pat00554
Figure pat00554

(공정 4) (Process 4)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-L-알라니네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-valyl-L-alaninate

상기 공정 3 에서 얻어진 화합물 (260 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (84 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 3 using the compound (260 mg) obtained in Step 3 above to obtain the title compound (84 mg).

Figure pat00555
Figure pat00555

(공정 5) (Process 5)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-알라닌아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-valyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10 -dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -7-yl] -9H-purin-2-yl} amino) ethyl] -L-alaninamide

(약물 링커 8) (drug linker 8)

실시예 8 공정 8-2 에서 얻어진 화합물 (5.3 ㎎) 과 상기 공정 4 에서 얻어진 화합물 (4.5 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 35 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (5.1 ㎎) 을 얻었다. Example 8 Using the compound (5.3 mg) obtained in Step 8-2 and the compound (4.5 mg) obtained in Step 4 above, a reaction was carried out in the same manner as in Example 23 Step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 25% - 35% (0 min - 30 min)] to obtain the title compound (5.1 mg).

Figure pat00556
Figure pat00556

실시예 69 : 약물 링커 9 의 합성 Example 69: Synthesis of Drug Linker 9

[합성 스킴][Synthesis Scheme]

[화학식 226][Formula 226]

Figure pat00557
Figure pat00557

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-프롤릴-L-이소류신 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-prolyl-L-isoleucine trifluoroacetate

시판 (Hangzhou Peptide Biochem) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-프롤릴-L-이소류신 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.02 g) 의 미정제체를 얻었다.The reaction was carried out in the same manner as in Example 21 step 1 using commercially available (Hangzhou Peptide Biochem) N-(tert-butoxycarbonyl)glycylglycyl-L-prolyl-L-isoleucine (1.00 g). and obtained a crude product of the title compound (1.02 g).

Figure pat00558
Figure pat00558

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-L-이소류신 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-prolyl-L-isoleucine

상기 공정 1 에서 얻어진 화합물 (1.02 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (993 ㎎) 을 얻었다. A reaction was carried out in the same manner as in Example 21 Step 2 using the compound (1.02 g) obtained in Step 1 above to obtain the title compound (993 mg).

Figure pat00559
Figure pat00559

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-L-이소류시네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-Prolyl-L-Isoleucinate

상기 공정 2 에서 얻어진 화합물 (300 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (287 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 3 using the compound (300 mg) obtained in Step 2 above to obtain the title compound (287 mg).

Figure pat00560
Figure pat00560

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-이소류신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycyl-L-prolyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2, 10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyn-7-yl]-9H-purin-2-yl}amino)ethyl]-L-isoleucinamide

(약물 링커 9) (drug linker 9)

실시예 8 공정 8-2 에서 얻어진 화합물 (51.8 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (36.9 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 35 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (62.5 ㎎) 을 얻었다. Example 8 Using the compound (51.8 mg) obtained in step 8-2 and the compound (36.9 mg) obtained in step 3 above, a reaction was conducted in the same manner as in Example 23 step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 35% (0 min - 30 min)] to obtain the title compound (62.5 mg).

MS(ESI)m/z : 1397(M-H)-.MS(ESI) m/z : 1397(MH) - .

실시예 70 : 약물 링커 10 의 합성 Example 70: Synthesis of Drug Linker 10

[합성 스킴][Synthesis Scheme]

[화학식 227][Formula 227]

Figure pat00561
Figure pat00561

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-페닐알라닐-L-메티오닌 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-phenylalanyl-L-methionine trifluoroacetate

시판 (Hangzhou Peptide Biochem) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-페닐알라닐-L-메티오닌 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.19 g) 의 미정제체를 얻었다.Using commercially available (Hangzhou Peptide Biochem) N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-L-methionine (1.00 g), the reaction was carried out in the same manner as in Example 21 Step 1. This was carried out to obtain a crude product of the title compound (1.19 g).

Figure pat00562
Figure pat00562

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-L-메티오닌 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-L-methionine

상기 공정 1 에서 얻어진 화합물 (1.03 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (652 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 2 using the compound (1.03 g) obtained in Step 1 above to obtain the title compound (652 mg).

Figure pat00563
Figure pat00563

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-L-메티오니네이트 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-L-methioninate

상기 공정 2 에서 얻어진 화합물 (201 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (95 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 3 using the compound (201 mg) obtained in Step 2 above to obtain the title compound (95 mg).

Figure pat00564
Figure pat00564

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-메티오닌아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2 ,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]-L-methioninamide

(약물 링커 10) (Drug Linker 10)

실시예 8 공정 8-2 에서 얻어진 화합물 (6.0 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (5.5 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (2.0 ㎎) 을 얻었다.Example 8 Using the compound (6.0 mg) obtained in step 8-2 and the compound (5.5 mg) obtained in step 3 above, a reaction was carried out in the same manner as in Example 23 step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)] to obtain the title compound (2.0 mg).

Figure pat00565
Figure pat00565

실시예 71 : 약물 링커 11 의 합성 Example 71: Synthesis of Drug Linker 11

[합성 스킴][Synthesis Scheme]

[화학식 228][Formula 228]

Figure pat00566
Figure pat00566

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-발릴-N5-카르바모일-L-오르니틴 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-valyl-N 5 -carbamoyl-L-ornithine trifluoroacetate

시판 (Hangzhou Peptide Biochem) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-발릴-N5-카르바모일-L-오르니틴 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.02 g) 의 미정제체를 얻었다.Using commercially available (Hangzhou Peptide Biochem) N-(tert-butoxycarbonyl)glycylglycyl-L-valyl-N 5 -carbamoyl-L-ornithine (1.00 g), Example 21 Step 1 The reaction was carried out in the same manner as above to obtain a crude product of the title compound (1.02 g).

Figure pat00567
Figure pat00567

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-N5-카르바모일-L-오르니틴 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N 5 -carba Moyl-L-Ornithine

상기 공정 1 에서 얻어진 화합물 (1.02 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (795 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 2 using the compound (1.02 g) obtained in Step 1 above to obtain the title compound (795 mg).

Figure pat00568
Figure pat00568

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-N5-카르바모일-L-오르니티네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-valyl-N 5 -carbamoyl-L-ornitinate

상기 공정 2 에서 얻어진 화합물 (300 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (177 ㎎) 을 얻었다.The reaction was carried out in the same manner as in Example 21 Step 3 using the compound (300 mg) obtained in Step 2 above to obtain the title compound (177 mg).

Figure pat00569
Figure pat00569

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-발릴-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-N5-카르바모일-L-오르니틴아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-valyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10 -dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -7-yl]-9H-purin-2-yl}amino)ethyl]-N 5 -carbamoyl-L-ornithinamide

(약물 링커 11) (drug linker 11)

실시예 8 공정 8-2 에서 얻어진 화합물 (5.0 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (3.8 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 35 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (3.0 ㎎) 을 얻었다. Example 8 Using the compound obtained in step 8-2 (5.0 mg) and the compound obtained in step 3 (3.8 mg), a reaction was carried out in the same manner as in Example 23 step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 35% (0 min - 30 min)] to obtain the title compound (3.0 mg).

MS(ESI)m/z : 1443(M-H)-.MS(ESI) m/z : 1443(MH) - .

실시예 72 : 약물 링커 12 의 합성 Example 72: Synthesis of Drug Linker 12

[합성 스킴][Synthesis Scheme]

[화학식 229][Formula 229]

Figure pat00570
Figure pat00570

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-페닐알라닐-N5-카르바모일-L-오르니틴 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-phenylalanyl-N 5 -carbamoyl-L-ornithine trifluoroacetate

시판 (Hangzhou Peptide Biochem) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-페닐알라닐-N5-카르바모일-L-오르니틴 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.05 g) 의 미정제체를 얻었다.Using commercially available (Hangzhou Peptide Biochem) N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N 5 -carbamoyl-L-ornithine (1.00 g), Example 21 The reaction was carried out in the same manner as in Step 1 to obtain a crude product of the title compound (1.05 g).

Figure pat00571
Figure pat00571

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N5-카르바모일-L-오르니틴 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N 5 - Carbamoyl-L-Ornithine

상기 공정 1 에서 얻어진 화합물 (1.05 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (550 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 21 Step 2 using the compound (1.05 g) obtained in Step 1 above to obtain the title compound (550 mg).

Figure pat00572
Figure pat00572

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N5-카르바모일-L-오르니티네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N 5 -carbamoyl-L-ornitinate

상기 공정 2 에서 얻어진 화합물 (225 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (226 ㎎) 을 얻었다.The reaction was carried out in the same manner as in Example 21 Step 3 using the compound (225 mg) obtained in Step 2 above to obtain the title compound (226 mg).

Figure pat00573
Figure pat00573

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-N5-카르바모일-L-오르니틴아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2 ,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]-N 5 -carbamoyl-L-ornithinamide

(약물 링커 12) (Drug Linker 12)

실시예 8 공정 8-2 에서 얻어진 화합물 (5.1 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (4.0 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 35 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (4.6 ㎎) 을 얻었다.Example 8 Using the compound (5.1 mg) obtained in Step 8-2 and the compound (4.0 mg) obtained in Step 3 above, a reaction was carried out in the same manner as in Example 23 Step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 35% (0 min - 30 min)] to obtain the title compound (4.6 mg).

Figure pat00574
Figure pat00574

실시예 73 : 약물 링커 13 의 합성 Example 73: Synthesis of Drug Linker 13

[합성 스킴][Synthesis Scheme]

[화학식 230][Formula 230]

Figure pat00575
Figure pat00575

(공정 1) (Process 1)

N-(아자니우밀아세틸)글리실-L-이소류실-N5-카르바모일-L-오르니틴 트리플루오로아세테이트 N-(Azaniumylacetyl)glycyl-L-isoleucyl-N 5 -carbamoyl-L-ornithine trifluoroacetate

시판 (Hangzhou Peptide Biochem) 되는 N-(tert-부톡시카르보닐)글리실글리실-L-이소류실-N5-카르바모일-L-오르니틴 (1.00 g) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.09 g) 의 미정제체를 얻었다.Using commercially available (Hangzhou Peptide Biochem) N-(tert-butoxycarbonyl)glycylglycyl-L-isoleucyl-N 5 -carbamoyl-L-ornithine (1.00 g), Example 21 process The reaction was carried out in the same manner as in 1 to obtain a crude product of the title compound (1.09 g).

Figure pat00576
Figure pat00576

(관측 가능한 피크만 기재) (Only observable peaks are listed)

(공정 2) (Process 2)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-이소류실-N5-카르바모일-L-오르니틴 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-isoleucyl-N 5 -carb Vamoyl-L-Ornithine

상기 공정 1 에서 얻어진 화합물 (1.08 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (524 ㎎) 을 얻었다.A reaction was carried out in the same manner as in Example 21 Step 2 using the compound (1.08 g) obtained in Step 1 above to obtain the title compound (524 mg).

Figure pat00577
Figure pat00577

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-이소류실-N5-카르바모일-L-오르니티네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-Isoleucyl-N 5 -Carbamoyl-L-Ornitinate

상기 공정 2 에서 얻어진 화합물 (250 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (224 ㎎) 을 얻었다.The reaction was carried out in the same manner as in Example 21 Step 3 using the compound (250 mg) obtained in Step 2 above to obtain the title compound (224 mg).

Figure pat00578
Figure pat00578

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-이소류실-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-N5-카르바모일-L-오르니틴아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycyl-L-isoleucyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2; 10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra decyn-7-yl]-9H-purin-2-yl}amino)ethyl]-N 5 -carbamoyl-L-ornithinamide

(약물 링커 13) (drug linker 13)

실시예 8 공정 8-2 에서 얻어진 화합물 (5.4 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (4.0 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 35 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (4.7 ㎎) 을 얻었다. Example 8 Using the compound (5.4 mg) obtained in step 8-2 and the compound (4.0 mg) obtained in step 3 above, a reaction was carried out in the same manner as in Example 23 step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 35% (0 min - 30 min)] to obtain the title compound (4.7 mg).

MS(ESI)m/z : 1457(M-H)-.MS(ESI) m/z : 1457(MH) - .

실시예 74 : 약물 링커 14 의 합성 Example 74: Synthesis of Drug Linker 14

[합성 스킴][Synthesis Scheme]

[화학식 231][Formula 231]

Figure pat00579
Figure pat00579

(공정 1) (Process 1)

N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리신 N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycine

시판 (도쿄 화성 공업) 되는 글리실글리신 (0.61 g) 을 사용하여, 실시예 21 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.08 g) 을 얻었다. Using commercially available (Tokyo Chemical Industry Co., Ltd.) glycylglycine (0.61 g), reaction was carried out in the same manner as in Example 21 Step 2 to obtain the title compound (1.08 g).

Figure pat00580
Figure pat00580

(공정 2) (Process 2)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리시네이트 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycinate

상기 공정 1 에서 얻어진 화합물 (1.07 g) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (942 ㎎) 을 얻었다. A reaction was carried out in the same manner as in Example 21 Step 3 using the compound (1.07 g) obtained in Step 1 above to obtain the title compound (942 mg).

Figure pat00581
Figure pat00581

(공정 3) (Process 3)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-L-류시네이트 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-Prolyl-L-Leucinate

상기 공정 2 에서 얻어진 화합물 (250 ㎎) 과 시판 (고쿠산 화학) 되는 L-프롤릴-L-류신 (166 ㎎) 의 N,N-디메틸포름아미드 (5.0 ㎖) 현탁액에, N,N-디이소프로필에틸아민 (0.25 ㎖) 을 첨가하고, 실온에서 2.5 시간 교반하였다. L-프롤릴-L-류신 (833 ㎎) 을 추가하여 밤새 교반하였다. 반응액을 감압 농축 후, 잔류물에 클로로포름 (50 ㎖) 과 10 ％ 시트르산 수용액 (10 ㎖) 을 첨가하고, 클로로포름으로 추출하였다. 유기층을 감압 농축하고, 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/(클로로포름 : 메탄올 : 물 = 7 : 3 : 1) 의 하층] 으로 조정제하였다. 미정제 생성물의 N,N-디메틸포름아미드 (5.0 ㎖) 용액에 N-하이드록시숙신이미드 (67 ㎎) 와 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (111 ㎎) 을 첨가하고, 실온에서 1.5 시간 교반하였다. 반응액을 감압 농축하고, 잔류물에 클로로포름 (40 ㎖) 과 물 (15 ㎖) 을 첨가하고, 클로로포름으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [클로로포름/메탄올], [아세트산에틸/메탄올] 로 차례로 정제하고, 표제 화합물 (127 ㎎) 을 얻었다.To a suspension of the compound (250 mg) obtained in step 2 above and commercially available (Kokusan Chemical) L-prolyl-L-leucine (166 mg) in N,N-dimethylformamide (5.0 ml), N,N-di Isopropylethylamine (0.25 ml) was added and stirred at room temperature for 2.5 hours. L-prolyl-L-leucine (833 mg) was added and stirred overnight. After concentrating the reaction solution under reduced pressure, chloroform (50 ml) and 10% citric acid aqueous solution (10 ml) were added to the residue, followed by extraction with chloroform. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [lower layer of chloroform/(chloroform:methanol:water = 7:3:1)]. N-hydroxysuccinimide (67 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (111 mg) were added to a solution of the crude product in N,N-dimethylformamide (5.0 ml). ) was added and stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and chloroform (40 ml) and water (15 ml) were added to the residue, followed by extraction with chloroform. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified sequentially by silica gel column chromatography [chloroform/methanol] and [ethyl acetate/methanol] to obtain the title compound (127 mg).

Figure pat00582
Figure pat00582

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-류신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycyl-L-prolyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2, 10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyn-7-yl]-9H-purin-2-yl}amino)ethyl]-L-leucinamide

(약물 링커 14) (Drug Linker 14)

실시예 8 공정 8-2 에서 얻어진 화합물 (6.9 ㎎) 과 상기 공정 3 에서 얻어진 화합물 (4.7 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (7.2 ㎎) 을 얻었다. Example 8 Using the compound (6.9 mg) obtained in Step 8-2 and the compound (4.7 mg) obtained in Step 3 above, a reaction was carried out in the same manner as in Example 23 Step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)] to obtain the title compound (7.2 mg).

MS(ESI)m/z : 1397(M-H)-.MS(ESI) m/z : 1397(MH) - .

실시예 75 : 약물 링커 15 의 합성 Example 75: Synthesis of Drug Linker 15

[합성 스킴][Synthesis Scheme]

[화학식 232][Formula 232]

Figure pat00583
Figure pat00583

(공정 1) (Process 1)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-알라닐-L-글루타미네이트 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-alanyl-L-glutaminate

시판 (와코 쥰야쿠 공업) 되는 L-알라닐-L-글루타민 (263 ㎎) 과 실시예 74 공정 2 에서 얻어진 화합물 (250 ㎎) 을 사용하여, 실시예 74 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (90 ㎎) 을 얻었다.Using commercially available (Wako Pure Chemical Industries) L-alanyl-L-glutamine (263 mg) and the compound obtained in Example 74 step 2 (250 mg), a reaction was carried out in the same manner as in Example 74 step 3, , which gave the title compound (90 mg).

Figure pat00584
Figure pat00584

(공정 2) (Process 2)

비스(N,N-디에틸에탄아미늄)N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-알라닐-N1-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-글루탐아미드 Bis(N,N-diethylethanenaminium)N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-alanyl-N 1 -[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2 ,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]-L-glutamamide

(약물 링커 15) (Drug Linker 15)

실시예 8 공정 8-2 에서 얻어진 화합물 (6.9 ㎎) 과 상기 공정 1 에서 얻어진 화합물 (4.7 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (3.9 ㎎) 을 얻었다. Example 8 Using the compound (6.9 mg) obtained in Step 8-2 and the compound (4.7 mg) obtained in Step 1 above, a reaction was carried out in the same manner as in Example 23 Step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)] to obtain the title compound (3.9 mg).

Figure pat00585
Figure pat00585

실시예 76 : 약물 링커 16 의 합성 Example 76: Synthesis of Drug Linker 16

[합성 스킴][Synthesis Scheme]

[화학식 233][Formula 233]

Figure pat00586
Figure pat00586

(공정 1) (Process 1)

2,5-디옥소피롤리딘-1-일 N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-L-프롤리네이트2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-Prolyl-L-Prolinate

시판 (Cool Pharm) 되는 1-(tert-부톡시카르보닐)-L-프롤릴-L-프롤린 (777 ㎎) 을 사용하여, 실시예 21 공정 1 과 동일한 수법으로 반응을 실시하고, 1-[(2S)-피롤리딘-1-이움-2-카르보닐]-L-프롤린 트리플루오로아세테이트의 미정제체를 얻었다. 이 미정제 생성물과 실시예 74 공정 2 에서 얻어진 화합물 (428 ㎎) 을 사용하여, 실시예 74 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (238 ㎎) 을 얻었다. Using commercially available (Cool Pharm) 1-(tert-butoxycarbonyl)-L-prolyl-L-proline (777 mg), the reaction was carried out in the same manner as in Example 21 Step 1, and 1-[ A crude product of (2S)-pyrrolidin-1-ium-2-carbonyl]-L-proline trifluoroacetate was obtained. Using this crude product and the compound (428 mg) obtained in Example 74 Step 2, a reaction was carried out in the same manner as in Example 74 Step 3 to obtain the title compound (238 mg).

Figure pat00587
Figure pat00587

(공정 2) (Process 2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-프롤릴-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]-L-프롤린아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl glycyl-L-prolyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2, 10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octa hydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra Decyn-7-yl]-9H-purin-2-yl}amino)ethyl]-L-prolinamide

(약물 링커 16) (drug linker 16)

실시예 8 공정 8-2 에서 얻어진 화합물 (6.9 ㎎) 과 상기 공정 1 에서 얻어진 화합물 (4.6 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 40 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (5.8 ㎎) 을 얻었다.Example 8 Using the compound (6.9 mg) obtained in Step 8-2 and the compound (4.6 mg) obtained in Step 1 above, a reaction was carried out in the same manner as in Example 23 Step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 20% - 40% (0 min - 30 min)] to obtain the title compound (5.8 mg).

MS(ESI)m/z : 1383(M＋H)＋.MS(ESI) m/z : 1383(M+H) + .

실시예 77 : 약물 링커 17 의 합성 Example 77: Synthesis of Drug Linker 17

[합성 스킴][Synthesis Scheme]

[화학식 234][Formula 234]

Figure pat00588
Figure pat00588

(공정 1) (Process 1)

[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메틸 아세테이트 [(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methyl acetate

시판 (SUNDIA) 되는 N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실글리신 (9.32 g) 을 사용하여, 실시예 67 공정 1 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (8.24 g) 을 얻었다.Using commercially available (SUNDIA) N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycylglycine (9.32 g), the reaction was carried out in the same manner as in Example 67 step 1, and the title compound (8.24 g) was obtained.

Figure pat00589
Figure pat00589

(공정 2) (Process 2)

2',3',5'-트리스-O-[tert-부틸(디메틸)실릴]-1-(2-하이드록시에틸)이노신 2',3',5'-tris-O-[tert-butyl(dimethyl)silyl]-1-(2-hydroxyethyl)inosine

이미 알려진 문헌 (Chem. Pharm. Bull. 1987, 35 (1), 72-79) 의 2',3',5'-트리스-O-[tert-부틸(디메틸)실릴]이노신 (31.3 g) 의 테트라하이드로푸란 (75 ㎖) - N,N-디메틸아세트아미드 (75 ㎖) 혼합 용액에, 2-브로모에탄올 (4.82 ㎖) 과 1,8-디아자비시클로[5.4.0]-7-운데센 (7.65 ㎖) 을 첨가하고, 실온에서 23 시간 교반하였다. 반응액에, 물과 아세트산에틸을 첨가하고, 아세트산에틸로 추출하였다. 유기층을 포화 식염수로 세정하고, 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (29.4 g) 을 얻었다.Of 2', 3', 5'-tris-O- [tert-butyl (dimethyl) silyl] inosine (31.3 g) of the previously known literature (Chem. Pharm. Bull. 1987, 35 (1), 72-79) To a mixed solution of tetrahydrofuran (75 mL) - N,N-dimethylacetamide (75 mL), 2-bromoethanol (4.82 mL) and 1,8-diazabicyclo[5.4.0]-7-undecene (7.65 mL) was added, and the mixture was stirred at room temperature for 23 hours. Water and ethyl acetate were added to the reaction mixture, and extraction was performed with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (29.4 g).

Figure pat00590
Figure pat00590

(공정 3) (Process 3)

2',3',5'-트리스-O-[tert-부틸(디메틸)실릴]-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신 2',3',5'-tris-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl) amino]methoxy}ethyl)inosine

상기 공정 2 에서 얻어진 화합물 (15.6 g) 의 톨루엔 (46.8 ㎖) 용액에 상기 공정 1 에서 얻어진 화합물 (10.4 g) 과 피리딘 (9.63 ㎖) 을 첨가하고, 110 ℃ 에서 12 시간 교반하였다. 반응액에 상기 공정 1 에서 얻어진 화합물 (3.46 g) 을 추가하고, 110 ℃ 에서 1 일간 교반하였다. 반응액에 포화 탄산수소나트륨 수용액과 디클로로메탄을 첨가하고, 디클로로메탄으로 추출하였다. 유기층을 무수 황산나트륨으로 건조 후, 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸] 로 정제하고, 표제 화합물 (20.6 g : 불순물 함유) 을 얻었다. The compound (10.4 g) and pyridine (9.63 ml) obtained in the above step 1 were added to a solution of the compound (15.6 g) obtained in the above step 2 in toluene (46.8 ml), and the mixture was stirred at 110°C for 12 hours. The compound (3.46 g) obtained in step 1 was added to the reaction solution, and the mixture was stirred at 110°C for 1 day. A saturated aqueous solution of sodium hydrogen carbonate and dichloromethane were added to the reaction mixture, followed by extraction with dichloromethane. After drying the organic layer over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate] to obtain the title compound (20.6 g: containing impurities).

MS(ESI)m/z : 885(M＋H)＋.MS(ESI) m/z : 885(M+H) + .

(공정 4) (Process 4)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신 5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methyl Toxy} ethyl) inosine

상기 공정 3 에서 얻어진 화합물 (20.6 g) 의 테트라하이드로푸란 (50 ㎖) 용액에 트리에틸아민3불화수소산염 (10 ㎖) 을 첨가하고, 실온에서 17 시간 교반하였다. 빙랭하, 반응액에 1 M 탄산수소트리에틸암모늄 용액 (50 ㎖) 과 트리에틸아민 (10 ㎖) 의 혼합액을 천천히 첨가한 후, 반응액을 감압 농축하였다. 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [물/아세토니트릴] 로 조정제 후, 동결 건조시켰다. 얻어진 미정제체를 피리딘으로 공비하고, 잔류물의 피리딘 (50 ㎖) 용액에, 4,4'-디메톡시트리틸클로라이드 (4.73 g) 를 0 ℃ 에서 첨가하고, 4 ℃ 에서 17 시간 교반하였다. 반응액에 메탄올 (2 ㎖) 을 첨가하고, 실온에서 15 분간 교반 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [헥산/아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (9.18 g : 불순물 함유) 을 얻었다.Triethylamine hydrofluoric acid salt (10 ml) was added to a solution of the compound (20.6 g) obtained in step 3 in tetrahydrofuran (50 ml), and the mixture was stirred at room temperature for 17 hours. Under ice-cooling, a mixture of 1 M triethylammonium hydrogen carbonate solution (50 ml) and triethylamine (10 ml) was slowly added to the reaction solution, and then the reaction solution was concentrated under reduced pressure. The residue was lyophilized after conditioning by C18 silica gel column chromatography [water/acetonitrile]. The resulting crude product was azeotroped with pyridine, and 4,4'-dimethoxytrityl chloride (4.73 g) was added to a solution of the residue in pyridine (50 ml) at 0°C, followed by stirring at 4°C for 17 hours. Methanol (2 ml) was added to the reaction solution, and after stirring at room temperature for 15 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane/ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (9.18 g: containing impurities).

Figure pat00591
Figure pat00591

(공정 5) (Process 5)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-( trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

상기 공정 4 에서 얻어진 화합물 (5.96 g) 을 사용하여, 실시예 5 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.33 g) 과 표제 화합물의 위치 이성체인 5'-O-[비스(4-메톡시페닐)(페닐)메틸]-2'-O-[tert-부틸(디메틸)실릴]-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신 (2.45 g) 을 얻었다.Using the compound (5.96 g) obtained in the above step 4, a reaction was carried out in the same manner as in Example 5 step 3 to obtain the title compound (2.33 g) and 5'-O-[bis(4) which is a positional isomer of the title compound. -methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl} This gave glycyl)amino]methoxy}ethyl)inosine (2.45 g).

Figure pat00592
Figure pat00592

위치 이성체 (2'-O-TBS 체) positional isomers (2'-O-TBS isomers)

Figure pat00593
Figure pat00593

(공정 6) (Process 6)

5'-O-[비스(4-메톡시페닐)(페닐)메틸]-3'-O-[tert-부틸(디메틸)실릴]-2'-O-{(2-시아노에톡시)[디(프로판-2-일)아미노]포스파닐}-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[ Di(propan-2-yl)amino]phosphanyl}-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

상기 공정 5 에서 얻어진 화합물 (2.33 g) 을 사용하여, 실시예 5 공정 4 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (2.72 g) 을 인 원자 상의 디아스테레오머 혼합물 (디아스테레오머비 = 6 : 4) 로서 얻었다.Using the compound (2.33 g) obtained in step 5 above, a reaction was carried out in the same manner as in step 4 of Example 5 to obtain the title compound (2.72 g) with a diastereomer mixture on a phosphorus atom (diastereomer ratio = 6:4). ) was obtained as

Figure pat00594
Figure pat00594

(공정 7) (Process 7)

실시예 44 공정 8 에서 얻어진 화합물 (2.15 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 상기 공정 6 에서 얻어진 화합물 (2.72 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (2.15 g) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (2.72 g) obtained in step 6 above, a reaction was carried out in the same manner as in step 8 of Example 1, and the obtained crude product was used as it was for the next reaction.

(공정 8) (Process 8)

2-(트리메틸실릴)에틸 (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-옥소-2-술파닐-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]아미노}-2-옥소에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cya noethoxy)-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl }ethoxy)methyl]amino}-2-oxoethyl)carbamate

상기 공정 7 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.47 g : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 7 above, a reaction was carried out in the same manner as in step 9 of Example 1 to obtain the title compound (1.47 g: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1278(M＋H)＋.MS(ESI) m/z : 1278(M+H) + .

(공정 9) (Process 9)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-7-[6-옥소-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)-1,6-디하이드로-9H-푸린-9-일]-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1 ,6-dihydro-9H-purin-9-yl] -14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo Spacyclotetradecine-2,10-bis(thiolate)

상기 공정 8 에서 얻어진 화합물 (1.47 g) 의 메탄올 (10 ㎖) - 테트라하이드로푸란 (10 ㎖) 혼합 용액에, 28 ％ 암모니아수 (10 ㎖) 를 첨가하고, 50 ℃ 에서 6 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 로 정제하고, 표제 화합물의 디아스테레오머 1 (204 ㎎ : 불순물 함유) 과 디아스테레오머 2 (205 ㎎ : 불순물 함유) 를 얻었다. 28% aqueous ammonia (10 ml) was added to a methanol (10 ml) -tetrahydrofuran (10 ml) mixed solution of the compound (1.47 g) obtained in step 8, and the mixture was stirred at 50°C for 6 hours. After concentration of the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] to obtain diastereomer 1 (204 mg: containing impurities) and diastereomer of the title compound. 2 (205 mg: containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1121(M＋H)＋. MS(ESI)m/z: 1121(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1121(M＋H)＋.MS(ESI)m/z: 1121(M+H) + .

(공정 10-1) (Process 10-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methyl Toxy] ethyl} -6-oxo-1,6-dihydro-9H-purin-9-yl) -16-hydroxy-2,10-dioxo-14- (6,7,8,9-tetrahydro -2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (디아스테레오머 1) (204 ㎎) 을 사용하여, 실시예 11 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 50 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (40.7 ㎎ : 불순물 함유) 을 얻었다. Using the compound (diastereomer 1) (204 mg) obtained in step 9 above, the reaction was carried out in the same manner as in step 9-1 of Example 11, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate] /acetonitrile] and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 50% (0 min - 40 min) ] to obtain the title compound (40.7 mg: containing impurities).

MS(ESI)m/z : 863(M＋H)＋.MS(ESI) m/z : 863(M+H) + .

(공정 10-2) (Process 10-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(티올레이트) Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methyl Toxy] ethyl} -6-oxo-1,6-dihydro-9H-purin-9-yl) -16-hydroxy-2,10-dioxo-14- (6,7,8,9-tetrahydro -2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(thiolate)

상기 공정 9 에서 얻어진 화합물 (디아스테레오머 2) (205 ㎎) 을 사용하여, 실시예 11 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 50 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (50.8 ㎎ : 불순물 함유) 을 얻었다. Using the compound (diastereomer 2) (205 mg) obtained in step 9 above, the reaction was carried out in the same manner as in step 9-1 of Example 11, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate] /acetonitrile] and preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 50% (0 min - 40 min) ] to obtain the title compound (50.8 mg: containing impurities).

MS(ESI)m/z : 863(M＋H)＋.MS(ESI) m/z : 863(M+H) + .

(공정 11-1) (Process 11-1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10 -dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(약물 링커 17a : 디아스테레오머 1) (Drug linker 17a: diastereomer 1)

상기 공정 10-1 에서 얻어진 화합물 (40.7 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 40 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (25.1 ㎎) 을 얻었다.Using the compound (40.7 mg) obtained in the above step 10-1, the reaction was carried out in the same manner as in Example 22 step 9-1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] , preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 20% - 45% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 40% -90% (0 min - 40 min)] to obtain the title compound (25.1 mg).

Figure pat00595
Figure pat00595

(공정 11-2) (Process 11-2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10 -dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro -2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine -7-yl] -6-oxo-6,9-dihydro-1H-purin-1-yl} ethoxy) methyl] glycinamide

(약물 링커 17b : 디아스테레오머 2) (Drug linker 17b: diastereomer 2)

상기 공정 10-2 에서 얻어진 화합물 (50.8 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 45 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (23.4 ㎎) 을 얻었다. Using the compound (50.8 mg) obtained in step 10-2 above, reaction was carried out in the same manner as in step 9-1 of Example 22, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] , preparative HPLC [10 mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 25% - 45% (0 min - 40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 45% -90% (0 min - 40 min)] to obtain the title compound (23.4 mg).

Figure pat00596
Figure pat00596

실시예 78 : 약물 링커 18 의 합성 Example 78: Synthesis of Drug Linker 18

[합성 스킴][Synthesis Scheme]

[화학식 235][Formula 235]

Figure pat00597
Figure pat00597

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10- Dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)glycinamide

(약물 링커 18) (drug linker 18)

실시예 45 공정 7-2 에서 얻어진 화합물 (10.0 ㎎) 과 실시예 21 공정 3 에서 얻어진 화합물 (8.8 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 50 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 45 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (10.9 ㎎) 을 얻었다. After reacting in the same manner as in Example 21 Step 4 using the compound (10.0 mg) obtained in Example 45 Step 7-2 and the compound (8.8 mg) obtained in Example 21 Step 3, C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% - 50% (0 min - 40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution/methanol, methanol: 45% - 90% (0 min - 40 min)] to obtain the title compound (10.9 mg).

Figure pat00598
Figure pat00598

실시예 79 : 약물 링커 19 의 합성 Example 79: Synthesis of Drug Linker 19

[합성 스킴][Synthesis Scheme]

[화학식 236][Formula 236]

Figure pat00599
Figure pat00599

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)아미노]-2-옥소에톡시}메틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy -2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2- 1) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadipo spacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)amino]-2-oxoethoxy}methyl)glycinamide

(약물 링커 19) (Drug Linker 19)

실시예 45 공정 7-2 에서 얻어진 화합물 (20.0 ㎎) 과 실시예 66 공정 1 에서 얻어진 화합물 (19.8 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 20 ％ - 45 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 30 ％ - 80 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (22.1 ㎎) 을 얻었다.After reacting in the same manner as in Example 21 step 4 using the compound (20.0 mg) obtained in step 7-2 of Example 45 and the compound (19.8 mg) obtained in step 1 of Example 66, C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% - 45% (0 min - 40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution/methanol, methanol: 30% - 80% (0 min - 40 min)] to obtain the title compound (22.1 mg).

Figure pat00600
Figure pat00600

실시예 80 : 약물 링커 20 의 합성 Example 80: Synthesis of Drug Linker 20

[합성 스킴][Synthesis Scheme]

[화학식 237][Formula 237]

Figure pat00601
Figure pat00601

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia- 2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfideoctahydro-2H ,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7 -yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)glycinamide

(약물 링커 20) (Drug Linker 20)

실시예 59 공정 4-2 에서 얻어진 화합물 (25.0 ㎎) 과 실시예 21 공정 3 에서 얻어진 화합물 (25.8 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 50 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 50 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (33.1 ㎎) 을 얻었다. Example 59 Using the compound (25.0 mg) obtained in step 4-2 and the compound (25.8 mg) obtained in Example 21 step 3 were reacted in the same manner as in Example 21 step 4, followed by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 30% - 50% (0 min - 40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution/methanol, methanol: 50% - 90% (0 min - 40 min)] to obtain the title compound (33.1 mg).

Figure pat00602
Figure pat00602

실시예 81 : 약물 링커 21 의 합성 Example 81: Synthesis of Drug Linker 21

[합성 스킴][Synthesis Scheme]

[화학식 238][Formula 238]

Figure pat00603
Figure pat00603

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에틸)아미노]-2-옥소에톡시}메틸)글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro -6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulf Pydooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)amino]-2-oxoethoxy}methyl)glycinamide

(약물 링커 21) (Drug Linker 21)

실시예 59 공정 4-2 에서 얻어진 화합물 (15.0 ㎎) 과 실시예 66 공정 1 에서 얻어진 화합물 (17.4 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 50 ％ (0 분 - 40 분)], 및 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 40 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물 (21.8 ㎎) 을 얻었다. Using the compound (15.0 mg) obtained in Example 59 step 4-2 and the compound (17.4 mg) obtained in Example 66 step 1, a reaction was carried out in the same manner as in Example 21 step 4, followed by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution/acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% - 50% (0 min - 40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution/methanol, methanol: 40% - 90% (0 min - 40 min)] to obtain the title compound (21.8 mg).

Figure pat00604
Figure pat00604

실시예 82 : 약물 링커 22 의 합성 Example 82: Synthesis of Drug Linker 22

[합성 스킴][Synthesis Scheme]

[화학식 239][Formula 239]

Figure pat00605
Figure pat00605

(공정 1) (Process 1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-디하이드로-6-티아-2,3,5-트리아자벤조[cd]아줄렌-2(7H)-일)-15-플루오로-16-하이드록시-2,10-디옥소-2,10-디술파이드옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro -6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulf Pydooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecin-7-yl]-9H-purin-2-yl}amino)ethyl]glycinamide

(약물 링커 22) (Drug Linker 22)

실시예 61 공정 4-2 에서 얻어진 화합물 (4.4 ㎎) 과 실시예 21 공정 3 에서 얻어진 화합물 (3.5 ㎎) 을 사용하여, 실시예 21 공정 4 와 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 25 ％ - 45 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (6.4 ㎎) 을 얻었다.After reacting in the same manner as in Example 21 step 4 using the compound (4.4 mg) obtained in Example 61 step 4-2 and the compound (3.5 mg) obtained in Example 21 step 3, preparative HPLC [10 mM Triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% - 45% (0 min - 30 min)] to obtain the title compound (6.4 mg).

Figure pat00606
Figure pat00606

실시예 83 : 약물 링커 23 의 합성 Example 83: Synthesis of Drug Linker 23

[합성 스킴][Synthesis Scheme]

[화학식 240][Formula 240]

Figure pat00607
Figure pat00607

(공정 1) (Process 1)

tert-부틸 N-[(벤질옥시)카르보닐]글리실글리실-L-페닐알라닐글리시네이트 tert-Butyl N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanylglycinate

시판 (BACHEM) 되는 N-[(벤질옥시)카르보닐]글리실글리실-L-페닐알라닌 (5.00 g) 과 글리신 tert-부틸에스테르염산염 (2.03 g) 의 N,N-디메틸포르미아미드 (50 ㎖) 용액에, 빙랭하, N,N-디이소프로필에틸아민 (4.11 ㎖) 과 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드염산염 (3.01 g) 을 첨가하고, 실온까지 승온하면서 밤새 교반하였다. 반응액을 클로로포름과 포화 탄산수소나트륨 수용액의 2 층에 따르고, 클로로포름으로 추출하였다. 유기층을 물과 포화 식염수로 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축 후, 톨루엔으로 2 회 공비하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (4.51 g) 을 얻었다.Commercially available (BACHEM) N,N-dimethylformiamide (50 ml ), N,N-diisopropylethylamine (4.11 ml) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (3.01 g) were added to the solution under ice-cooling, and the temperature was raised to room temperature. while stirring overnight. The reaction solution was poured into two layers of chloroform and saturated aqueous sodium hydrogen carbonate solution, and extracted with chloroform. The organic layer was washed with water and brine, and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was azeotroped twice with toluene after concentration under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (4.51 g).

Figure pat00608
Figure pat00608

(공정 2) (Process 2)

tert-부틸 글리실글리실-L-페닐알라닐글리시네이트 tert-Butyl glycylglycyl-L-phenylalanylglycinate

상기 공정 1 에서 얻어진 화합물 (4.51 g) 의 메탄올 (20 ㎖) - 디클로로메탄 (80 ㎖) 혼합액에, 10 ％ 팔라듐탄소 (M) wet (750 ㎎) 를 첨가하고, 수소 분위기하, 실온에서 밤새 교반하였다. 10 ％ 팔라듐탄소 (M) wet (1.5 g) 를 추가하고, 수소 분위기하, 실온에서 밤새 교반하였다. 반응액을 셀라이트로 여과하고, 여과액을 감압 농축하였다. 잔류물을 아미노 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (3.18 g) 을 얻었다. 10% palladium carbon (M) wet (750 mg) was added to a mixture of methanol (20 ml) and dichloromethane (80 ml) of the compound (4.51 g) obtained in step 1 above, and stirred overnight at room temperature under a hydrogen atmosphere. did 10% palladium carbon (M) wet (1.5 g) was added, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography [dichloromethane/methanol] to give the title compound (3.18 g).

Figure pat00609
Figure pat00609

(공정 3) (Process 3)

tert-부틸 N-[6-(2,5-디옥소-2,5-디하이드로-1H-피롤-1-일)헥사노일]글리실글리실-L-페닐알라닐글리시네이트 tert-Butyl N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanylglycinate

상기 공정 2 에서 얻어진 화합물 (2.65 g) 과 시판 (도쿄 화성 공업) 되는 6-말레이미드헥산산 N-숙신이미딜 (2.20 g) 을 N,N-디메틸포르미아미드 (20 ㎖) 에 용해하고, 실온에서 7 시간 교반하였다. 반응액을 감압 농축 후, 잔류물을 디클로로메탄과 물의 2 층에 따르고, 디클로로메탄으로 추출하였다. 유기층을 물로 3 회, 포화 식염수로 1 회 세정 후, 무수 황산나트륨으로 건조시켰다. 건조제를 여과 제거하고, 여과액을 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (3.52 g) 을 얻었다.The compound (2.65 g) obtained in step 2 above and N-succinimidyl 6-maleimidehexanoate (2.20 g) commercially available (Tokyo Chemical Industry) were dissolved in N,N-dimethylformiamide (20 ml), It was stirred at room temperature for 7 hours. After concentration of the reaction solution under reduced pressure, the residue was poured into two layers of dichloromethane and water and extracted with dichloromethane. The organic layer was washed three times with water and once with saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (3.52 g).

Figure pat00610
Figure pat00610

(공정 4) (Process 4)

N-[6-(2,5-디옥소-2,5-디하이드로-1H-피롤-1-일)헥사노일]글리실글리실-L-페닐알라닐글리신 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanylglycine

상기 공정 3 에서 얻어진 화합물 (1.00 g) 의 디클로로메탄 (9.0 ㎖) 용액에, 0 ℃ 에서 트리플루오로아세트산 (4.5 ㎖) 을 첨가하고, 실온에서 2.5 시간 교반하였다. 반응액을 감압 농축 후, 톨루엔으로 2 회 공비하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [디클로로메탄/메탄올] 로 정제하고, 표제 화합물 (534 ㎎) 을 얻었다. Trifluoroacetic acid (4.5 ml) was added to a solution of the compound (1.00 g) obtained in step 3 in dichloromethane (9.0 ml) at 0°C, and the mixture was stirred at room temperature for 2.5 hours. After concentration under reduced pressure, the reaction solution was azeotroped twice with toluene. The residue was purified by silica gel column chromatography [dichloromethane/methanol] to give the title compound (534 mg).

Figure pat00611
Figure pat00611

(공정 5) (Process 5)

2,5-디옥소피롤리딘-1-일 N-[6-(2,5-디옥소-2,5-디하이드로-1H-피롤-1-일)헥사노일]글리실글리실-L-페닐알라닐글리시네이트 2,5-dioxopyrrolidin-1-yl N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L- Phenylalanylglycinate

상기 공정 4 에서 얻어진 화합물 (462 ㎎) 을 사용하여, 실시예 21 공정 3 과 동일한 수법으로 반응을 실시하고, 표제 화합물 (258 ㎎) 을 얻었다. The reaction was carried out in the same manner as in Example 21 Step 3 using the compound (462 mg) obtained in Step 4 above to obtain the title compound (258 mg).

Figure pat00612
Figure pat00612

(공정 6) (Process 6)

비스(N,N-디에틸에탄아미늄) N-[6-(2,5-디옥소-2,5-디하이드로-1H-피롤-1-일)헥사노일]글리실글리실-L-페닐알라닐-N-[2-({6-아미노-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-디하이드록시-2,10-디옥소-2,10-디술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-9H-푸린-2-일}아미노)에틸]글리신아미드 Bis(N,N-diethylethanelaminium) N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L- Phenylalanyl-N-[2-({6-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo -2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H; 10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7- yl]-9H-purin-2-yl}amino)ethyl]glycinamide

(약물 링커 23) (drug linker 23)

실시예 8 공정 8-2 에서 얻어진 화합물 (9.5 ㎎) 과 상기 공정 5 에서 얻어진 화합물 (7.5 ㎎) 을 사용하여, 실시예 23 공정 1 과 동일한 수법으로 반응을 실시한 후, 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 30 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (7.8 ㎎) 을 얻었다. Example 8 Using the compound (9.5 mg) obtained in step 8-2 and the compound (7.5 mg) obtained in step 5, a reaction was carried out in the same manner as in Example 23 step 1, followed by preparative HPLC [10 mM triacetic acid Ethylammonium aqueous solution/acetonitrile, acetonitrile: 10% - 30% (0 min - 30 min)] to obtain the title compound (7.8 mg).

Figure pat00613
Figure pat00613

실시예 84 : 당사슬 리모델링 항체 3 의 합성 Example 84: Synthesis of sugar chain remodeling antibody 3

항HER2 항체 2 - [SG-(N3)2]2 의 조제 Preparation of anti-HER2 antibody 2 - [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 241][Formula 241]

Figure pat00614
Figure pat00614

(공정 1) (Process 1)

(Fucα1,6)GlcNAc-항HER2 항체 2 의 조제 Preparation of (Fucα1,6)GlcNAc-anti-HER2 antibody 2

시판되는 PERJETA (등록상표) 점적정주 (點滴靜注) 420 ㎎/14 ㎖ (츄가이 제약) (3.5 ㎖) 를 공통 조작 C 에 따라서 버퍼 교환하고, 인산 완충 생리 식염수 용액 (6.0 ㎖, 15.39 ㎎/㎖, pH 6.0) 을 얻었다. 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (12.96 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.Commercially available PERJETA (registered trademark) drip infusion (點滴靜注) 420 mg/14 ml (Chugai Pharmaceutical) (3.5 ml) was buffer-exchanged according to common procedure C, followed by phosphate buffered saline solution (6.0 ml, 15.39 mg/ml). ml, pH 6.0) was obtained. The same operation as in Example 25 step 1 was performed to obtain a 20 mM phosphate buffer solution (12.96 mg/ml, 7.5 ml, pH 6.0) of the title antibody.

(공정 2) (Process 2)

항HER2 항체 2 - [SG-(N3)2]2 의 조제 Preparation of anti-HER2 antibody 2 - [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (12.96 ㎎/㎖, 7.5 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (22.5 ㎎) 를 사용하여, 실시예 25 공정 2 과 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.21 ㎎/㎖, 9.0 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (12.96 mg/mL, 7.5 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (22.5 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (10.21 mg/ml, 9.0 ml, pH 6.0) of the title antibody.

실시예 85 : 당사슬 리모델링 항체 4 의 합성 Example 85: Synthesis of sugar chain remodeling antibody 4

개변 항HER2 항체 2 - [SG-(N3)2]2 의 조제 Preparation of modified anti-HER2 antibody 2-[SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 242][Formula 242]

Figure pat00615
Figure pat00615

(공정 1) (Process 1)

(Fucα1,6)GlcNAc - 개변 항HER2 항체 2 의 조제 Preparation of (Fucα1,6)GlcNAc-modified anti-HER2 antibody 2

참고예 5 에 따라서 조제한 개변 항HER2 항체 2 의 인산 완충 생리 식염수 용액 (24 ㎖, 12.25 ㎎/㎖, pH 6.0) 을 사용하여, 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (20.86 ㎎/㎖, 12.5 ㎖, pH 6.0) 을 얻었다.Using the modified anti-HER2 antibody 2 prepared in Reference Example 5 in phosphate-buffered physiological saline solution (24 ml, 12.25 mg/ml, pH 6.0), the same operation as in Example 25 Step 1 was performed, and 20 mM of the title antibody was added. A phosphate buffer solution (20.86 mg/ml, 12.5 ml, pH 6.0) was obtained.

(공정 2) (Process 2)

개변 항HER2 항체 2 - [SG-(N3)2]2 의 조제 Preparation of modified anti-HER2 antibody 2-[SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (20.86 ㎎/㎖, 12.5 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (52 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.87 ㎎/㎖, 22 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (20.86 mg/mL, 12.5 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (52 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (10.87 mg/ml, 22 ml, pH 6.0) of the title antibody.

실시예 86 : 당사슬 리모델링 항체 5 의 합성 Example 86: Synthesis of sugar chain remodeling antibody 5

항CD33 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-CD33 antibody - [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 243][Formula 243]

Figure pat00616
Figure pat00616

(공정 1) (Process 1)

(Fucα1,6)GlcNAc-항CD33 항체의 조제 Preparation of (Fucα1,6)GlcNAc-anti-CD33 antibody

참고예 6 에 따라서 조제한 항CD33 항체의 인산 완충 생리 식염수 용액 (9.0 ㎖, 11.56 ㎎/㎖, pH 6.0) 을 사용하여, 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (11.62 ㎎/㎖, 8 ㎖, pH 6.0) 을 얻었다.Using the anti-CD33 antibody phosphate-buffered physiological saline solution (9.0 ml, 11.56 mg/ml, pH 6.0) prepared according to Reference Example 6, the same operation as in Example 25 Step 1 was performed, and the title antibody in 20 mM phosphate buffer A solution (11.62 mg/mL, 8 mL, pH 6.0) was obtained.

(공정 2) (Process 2)

항CD33 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-CD33 antibody - [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (11.62 ㎎/㎖, 8 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (21.5 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.01 ㎎/㎖, 8 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (11.62 mg/mL, 8 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (21.5 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (10.01 mg/ml, 8 ml, pH 6.0) of the title antibody.

실시예 87 : 당사슬 리모델링 항체 6 의 합성 Example 87: Synthesis of sugar chain remodeling antibody 6

항EphA2 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-EphA2 antibody - [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 244][Formula 244]

Figure pat00617
Figure pat00617

(공정 1) (Process 1)

(Fucα1,6)GlcNAc-항EphA2 항체의 조제 Preparation of (Fucα1,6)GlcNAc-anti-EphA2 antibody

참고예 7 에 따라서 조제한 항EphA2 항체의 인산 완충 생리 식염수 용액 (8.0 ㎖, 12.83 ㎎/㎖, pH 6.0) 을 사용하여, 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (13.51 ㎎/㎖, 7 ㎖, pH 6.0) 을 얻었다.Using the anti-EphA2 antibody prepared in Reference Example 7 in phosphate-buffered physiological saline solution (8.0 ml, 12.83 mg/ml, pH 6.0), the same operation as in Example 25 Step 1 was performed, and the title antibody in 20 mM phosphate buffer A solution (13.51 mg/mL, 7 mL, pH 6.0) was obtained.

(공정 2) (Process 2)

항EphA2 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-EphA2 antibody - [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (13.51 ㎎/㎖, 7 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (21.7 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (8.91 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (13.51 mg/mL, 7 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (21.7 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (8.91 mg/ml, 7.5 ml, pH 6.0) of the title antibody.

실시예 88 : 당사슬 리모델링 항체 7 의 합성 Example 88: Synthesis of sugar chain remodeling antibody 7

항CDH6 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-CDH6 antibody - [SG-(N 3 ) 2 ] 2

[합성 스킴][Synthesis Scheme]

[화학식 245][Formula 245]

Figure pat00618
Figure pat00618

(공정 1) (Process 1)

(Fucα1,6)GlcNAc-항CDH6 항체의 조제 Preparation of (Fucα1,6)GlcNAc-anti-CDH6 antibody

참고예 8 에 따라서 조제한 항CDH6 항체의 HBSor (25 mM 히스티딘/5 ％ 소르비톨, pH = 6.0) 완충액 (5.0 ㎖, 20.0 ㎎/㎖) 을 공통 조작 C 에 따라서, 인산 완충 생리 식염수 용액 (pH = 6.0) 으로 버퍼 교환 후, 실시예 25 공정 1 과 동일한 조작을 실시하고, 표제 항체의 20 mM 인산 완충 용액 (9.58 ㎎/㎖, 9.0 ㎖, pH 6.0) 을 얻었다.HBSor (25 mM histidine/5% sorbitol, pH = 6.0) buffer solution (5.0 ml, 20.0 mg/ml) of the anti-CDH6 antibody prepared according to Reference Example 8 was mixed with a phosphate buffered physiological saline solution (pH = 6.0) according to common procedure C. ), the same operation as in Example 25 step 1 was performed after buffer exchange to obtain a 20 mM phosphate buffer solution (9.58 mg/ml, 9.0 ml, pH 6.0) of the title antibody.

(공정 2) (Process 2)

항CDH6 항체 - [SG-(N3)2]2 의 조제 Preparation of anti-CDH6 antibody - [SG-(N 3 ) 2 ] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (9.58 ㎎/㎖, 9.0 ㎖, pH 6.0) 과 [N3-PEG(3)]2-SG(10)Ox (24.5 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.69 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.Using a 20 mM phosphate buffer solution (9.58 mg/mL, 9.0 mL, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)] 2 -SG(10)Ox (24.5 mg), The same operation as in Example 25 step 2 was performed to obtain a phosphate-buffered physiological saline solution (10.69 mg/ml, 7.5 ml, pH 6.0) of the title antibody.

실시예 89 : 당사슬 리모델링 항체 8 의 합성 Example 89: Synthesis of sugar chain remodeling antibody 8

개변 항HER2 항체 2 - [MSG1-(N3)]2 의 조제 Preparation of modified anti-HER2 antibody 2-[MSG1-(N 3 )] 2

[합성 스킴][Synthesis Scheme]

[화학식 246][Formula 246]

Figure pat00619
Figure pat00619

(공정 1) (Process 1)

(Fucα1,6)GlcNAc - 개변 항HER2 항체 2 의 조제 Preparation of (Fucα1,6)GlcNAc-modified anti-HER2 antibody 2

실시예 85 공정 1 과 동일한 반응을 실시하고, 원료 항체의 완충 용액 (10 ㎖, 12.25 ㎎/㎖, pH 6.0) 으로부터, 표제 항체의 20 mM 인산 완충 용액 (14.84 ㎎/㎖, 7.5 ㎖, pH 6.0) 을 얻었다.The same reaction as in Example 85 step 1 was carried out, and a 20 mM phosphate buffer solution (14.84 mg/ml, 7.5 ml, pH 6.0) of the title antibody was added from the buffer solution (10 ml, 12.25 mg/ml, pH 6.0) of the starting antibody. ) was obtained.

(공정 2) (Process 2)

개변 항HER2 항체 2 - [MSG1-(N3)]2 의 조제 Preparation of modified anti-HER2 antibody 2-[MSG1-(N 3 )] 2

상기 공정 1 에서 얻어진 항체의 20 mM 인산 완충 용액 (14.84 ㎎/㎖, 7.5 ㎖, pH 6.0) 과 [N3-PEG(3)]-MSG1(9)-Ox (WO2018/003983 의 화합물 1-11) (19 ㎎) 를 사용하여, 실시예 25 공정 2 와 동일한 조작을 실시하고, 표제 항체의 인산 완충 생리 식염수 용액 (10.48 ㎎/㎖, 9.25 ㎖, pH 6.0) 을 얻었다.A 20 mM phosphate buffer solution (14.84 mg/ml, 7.5 ml, pH 6.0) of the antibody obtained in step 1 above and [N 3 -PEG(3)]-MSG1(9)-Ox (Compound 1-11 of WO2018/003983) ) (19 mg) was used to obtain a phosphate-buffered physiological saline solution (10.48 mg/mL, 9.25 mL, pH 6.0) of the title antibody by performing the same operation as in Example 25 step 2.

실시예 90 : 항체 약물 콘쥬게이트 5 의 합성 (항HER2 항체 - CDN 콘쥬게이트 4 의 합성) Example 90: Synthesis of Antibody Drug Conjugate 5 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 4)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.97 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 1 의 디메틸술폭시드 용액 (10 mM, 0.091 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.159 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.97 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 1 dimethylsulfoxide solution (10 mM, 0.091 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.159 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.49 ㎎/㎖ Antibody concentration: 0.49 mg/ml

항체 수량 : 1.71 ㎎ (31 ％) Antibody quantity: 1.71 mg (31%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 91 : 항체 약물 콘쥬게이트 6 의 합성 (항HER2 항체 - CDN 콘쥬게이트 5 의 합성) Example 91: Synthesis of Antibody Drug Conjugate 6 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 5)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.97 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2a 의 디메틸술폭시드 용액 (10 mM, 0.091 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.159 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.97 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2a dimethylsulfoxide solution (10 mM, 0.091 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.159 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.91 ㎎/㎖ Antibody concentration: 0.91 mg/ml

항체 수량 : 3.17 ㎎ (58 ％) Antibody quantity: 3.17 mg (58%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 92 : 항체 약물 콘쥬게이트 7 의 합성 (항HER2 항체 - CDN 콘쥬게이트 6 의 합성) Example 92: Synthesis of Antibody Drug Conjugate 7 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 6)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 4.00 ㎖) 을 프로필렌글리콜 (2.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 2b 의 디메틸술폭시드 용액 (10 mM, 0.707 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (1.293 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (24.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 4.00 ml) was diluted with propylene glycol (2.00 ml). To this solution, a mixture of drug linker 2b dimethylsulfoxide solution (10 mM, 0.707 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (1.293 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (24.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.96 ㎎/㎖ Antibody concentration: 0.96 mg/ml

항체 수량 : 23.57 ㎎ (59 ％) Antibody quantity: 23.57 mg (59%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 93 : 항체 약물 콘쥬게이트 8 의 합성 (항HER2 항체 - CDN 콘쥬게이트 7 의 합성) Example 93: Synthesis of Antibody Drug Conjugate 8 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 7)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 4.00 ㎖) 을 프로필렌글리콜 (2.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 5 의 디메틸술폭시드 용액 (10 mM, 0.707 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (1.293 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (24.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 4.00 ml) was diluted with propylene glycol (2.00 ml). To this solution, a mixture of drug linker 5 dimethylsulfoxide solution (10 mM, 0.707 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (1.293 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (24.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.10 ㎎/㎖ Antibody concentration: 1.10 mg/ml

항체 수량 : 26.86 ㎎ (67 ％) Antibody quantity: 26.86 mg (67%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 94 : 항체 약물 콘쥬게이트 9 의 합성 (항HER2 항체 - CDN 콘쥬게이트 8 의 합성) Example 94: Synthesis of Antibody Drug Conjugate 9 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 8)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 4.00 ㎖) 을 프로필렌글리콜 (2.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 6 의 디메틸술폭시드 용액 (10 mM, 0.707 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (1.293 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (24.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 4.00 ml) was diluted with propylene glycol (2.00 ml). To this solution, a mixture of drug linker 6 dimethylsulfoxide solution (10 mM, 0.707 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (1.293 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (24.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.34 ㎎/㎖ Antibody concentration: 1.34 mg/ml

항체 수량 : 32.92 ㎎ (82 ％) Antibody quantity: 32.92 mg (82%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 95 : 항체 약물 콘쥬게이트 10 의 합성 (항LPS 항체 - CDN 콘쥬게이트 2 의 합성) Example 95: Synthesis of Antibody Drug Conjugate 10 (Synthesis of Anti-LPS Antibody-CDN Conjugate 2)

당사슬 리모델링 항체 2 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.55 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2a 의 디메틸술폭시드 용액 (10 mM, 0.087 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.163 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 2 in phosphate buffered saline (pH 6.0) (10.55 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2a dimethylsulfoxide solution (10 mM, 0.087 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.163 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.88 ㎎/㎖ Antibody concentration: 0.88 mg/ml

항체 수량 : 3.08 ㎎ (62 ％) Antibody quantity: 3.08 mg (62%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 96 : 항체 약물 콘쥬게이트 11 의 합성 (항EphA2 항체 - CDN 콘쥬게이트 1 의 합성) Example 96: Synthesis of antibody drug conjugate 11 (anti-EphA2 antibody - synthesis of CDN conjugate 1)

당사슬 리모델링 항체 6 의 인산 완충 생리 식염수 (pH 6.0) 용액 (8.91 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2b 의 디메틸술폭시드 용액 (10 mM, 0.073 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.177 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 6 in phosphate buffered saline (pH 6.0) (8.91 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2b dimethylsulfoxide solution (10 mM, 0.073 ml, 24 equivalents per antibody molecule) and propylene glycol (0.177 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 F 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and F, and the following results were obtained.

항체 농도 : 0.60 ㎎/㎖ Antibody concentration: 0.60 mg/ml

항체 수량 : 2.10 ㎎ (47 ％) Antibody quantity: 2.10 mg (47%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 97 : 항체 약물 콘쥬게이트 12 의 합성 (항CD33 항체 - CDN 콘쥬게이트 1 의 합성) Example 97: Synthesis of Antibody Drug Conjugate 12 (Synthesis of Anti-CD33 Antibody - CDN Conjugate 1)

당사슬 리모델링 항체 5 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.01 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2b 의 디메틸술폭시드 용액 (10 mM, 0.083 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.167 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 5 in phosphate buffered saline (pH 6.0) (10.01 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2b dimethylsulfoxide solution (10 mM, 0.083 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.167 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.73 ㎎/㎖ Antibody concentration: 0.73 mg/ml

항체 수량 : 2.57 ㎎ (51 ％) Antibody quantity: 2.57 mg (51%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 98 : 항체 약물 콘쥬게이트 13 의 합성 (항CDH6 항체 - CDN 콘쥬게이트 1 의 합성) Example 98: Synthesis of Antibody Drug Conjugate 13 (Synthesis of Anti-CDH6 Antibody-CDN Conjugate 1)

당사슬 리모델링 항체 7 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.69 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 2b 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 7 in phosphate buffered saline (pH 6.0) (10.69 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 2b dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.96 ㎎/㎖ Antibody concentration: 0.96 mg/ml

항체 수량 : 3.37 ㎎ (63 ％) Antibody quantity: 3.37 mg (63%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 99 : 항체 약물 콘쥬게이트 14 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 1 의 합성) Example 99: Synthesis of Antibody Drug Conjugate 14 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 1)

당사슬 리모델링 항체 3 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.21 ㎎/㎖, 1.50 ㎖) 을 프로필렌글리콜 (0.750 ㎖) 로 희석하였다. 이 용액에 약물 링커 3 의 디메틸술폭시드 용액 (10 mM, 0.253 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.497 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (9.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 3 in phosphate buffered saline (pH 6.0) (10.21 mg/ml, 1.50 ml) was diluted with propylene glycol (0.750 ml). To this solution, a mixture of drug linker 3 dimethylsulfoxide solution (10 mM, 0.253 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.497 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (9.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.02 ㎎/㎖ Antibody concentration: 1.02 mg/ml

항체 수량 : 9.73 ㎎ (64 ％) Antibody quantity: 9.73 mg (64%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 100 : 항체 약물 콘쥬게이트 15 의 합성 (항HER2 항체 - CDN 콘쥬게이트 9 의 합성) Example 100: Synthesis of Antibody Drug Conjugate 15 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 9)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 8 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 8 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.93 ㎎/㎖ Antibody concentration: 0.93 mg/ml

항체 수량 : 3.24 ㎎ (61 ％) Antibody quantity: 3.24 mg (61%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 101 : 항체 약물 콘쥬게이트 16 의 합성 (항HER2 항체 - CDN 콘쥬게이트 10 의 합성) Example 101: Synthesis of Antibody Drug Conjugate 16 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 10)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.35 ㎎/㎖, 13.50 ㎖) 을 프로필렌글리콜 (6.750 ㎖) 로 희석하였다. 이 용액에 약물 링커 9 의 디메틸술폭시드 용액 (10 mM, 2.310 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (4.440 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (74.3 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.35 mg/ml, 13.50 ml) was diluted with propylene glycol (6.750 ml). To this solution, a mixture of drug linker 9 dimethylsulfoxide solution (10 mM, 2.310 ml, 24 equivalents per antibody molecule) and propylene glycol (4.440 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (74.3 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.36 ㎎/㎖ Antibody concentration: 1.36 mg/ml

항체 수량 : 100.8 ㎎ (72 ％) Antibody quantity: 100.8 mg (72%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 102 : 항체 약물 콘쥬게이트 17 의 합성 (항HER2 항체 - CDN 콘쥬게이트 11 의 합성) Example 102: Synthesis of Antibody Drug Conjugate 17 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 11)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 10 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 10 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.63 ㎎/㎖ Antibody concentration: 0.63 mg/ml

항체 수량 : 2.22 ㎎ (42 ％) Antibody quantity: 2.22 mg (42%)

약물 평균 결합수 : 3.5Average number of drug bonds: 3.5

실시예 103 : 항체 약물 콘쥬게이트 18 의 합성 (항HER2 항체 - CDN 콘쥬게이트 12 의 합성) Example 103: Synthesis of Antibody Drug Conjugate 18 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 12)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 11 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 11 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.94 ㎎/㎖ Antibody concentration: 0.94 mg/ml

항체 수량 : 3.29 ㎎ (62 ％) Antibody quantity: 3.29 mg (62%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 104 : 항체 약물 콘쥬게이트 19 의 합성 (항HER2 항체 - CDN 콘쥬게이트 13 의 합성) Example 104: Synthesis of Antibody Drug Conjugate 19 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 13)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 12 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 12 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.92 ㎎/㎖ Antibody concentration: 0.92 mg/ml

항체 수량 : 3.20 ㎎ (60 ％) Antibody quantity: 3.20 mg (60%)

약물 평균 결합수 : 3.5Average number of drug bonds: 3.5

실시예 105 : 항체 약물 콘쥬게이트 20 의 합성 (항HER2 항체 - CDN 콘쥬게이트 14 의 합성) Example 105: Synthesis of Antibody Drug Conjugate 20 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 14)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 13 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 13 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.01 ㎎/㎖ Antibody concentration: 1.01 mg/ml

항체 수량 : 3.52 ㎎ (66 ％) Antibody quantity: 3.52 mg (66%)

약물 평균 결합수 : 3.5Average number of drug bonds: 3.5

실시예 106 : 항체 약물 콘쥬게이트 21 의 합성 (항HER2 항체 - CDN 콘쥬게이트 15 의 합성) Example 106: Synthesis of Antibody Drug Conjugate 21 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 15)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 14 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 14 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.78 ㎎/㎖ Antibody concentration: 0.78 mg/ml

항체 수량 : 2.74 ㎎ (51 ％) Antibody quantity: 2.74 mg (51%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 107 : 항체 약물 콘쥬게이트 22 의 합성 (항HER2 항체 - CDN 콘쥬게이트 16 의 합성) Example 107: Synthesis of antibody drug conjugate 22 (anti-HER2 antibody - synthesis of CDN conjugate 16)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 15 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 15 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.32 ㎎/㎖ Antibody concentration: 1.32 mg/ml

항체 수량 : 4.61 ㎎ (86 ％) Antibody quantity: 4.61 mg (86%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 108 : 항체 약물 콘쥬게이트 23 의 합성 (항HER2 항체 - CDN 콘쥬게이트 17 의 합성) Example 108: Synthesis of Antibody Drug Conjugate 23 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 17)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 16 의 디메틸술폭시드 용액 (10 mM, 0.088 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.162 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 16 dimethylsulfoxide solution (10 mM, 0.088 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.162 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.93 ㎎/㎖ Antibody concentration: 0.93 mg/ml

항체 수량 : 3.25 ㎎ (61 ％) Antibody quantity: 3.25 mg (61%)

약물 평균 결합수 : 3.6Average number of drug bonds: 3.6

실시예 109 : 항체 약물 콘쥬게이트 24 의 합성 (항HER2 항체 - CDN 콘쥬게이트 18 의 합성) Example 109: Synthesis of antibody drug conjugate 24 (anti-HER2 antibody - synthesis of CDN conjugate 18)

당사슬 리모델링 항체 1 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.70 ㎎/㎖, 4.00 ㎖) 을 프로필렌글리콜 (2.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 7 의 디메틸술폭시드 용액 (10 mM, 0.707 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (1.293 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (24.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 1 in phosphate buffered saline (pH 6.0) (10.70 mg/ml, 4.00 ml) was diluted with propylene glycol (2.00 ml). To this solution, a mixture of drug linker 7 dimethylsulfoxide solution (10 mM, 0.707 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (1.293 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (24.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.33 ㎎/㎖ Antibody concentration: 1.33 mg/ml

항체 수량 : 32.64 ㎎ (82 ％) Antibody quantity: 32.64 mg (82%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 110 : 항체 약물 콘쥬게이트 25 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 2 의 합성) Example 110: Synthesis of Antibody Drug Conjugate 25 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 2)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.87 ㎎/㎖, 8.00 ㎖) 을 프로필렌글리콜 (4.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 17a 의 디메틸술폭시드 용액 (10 mM, 1.079 ㎖, 항체 1 분자에 대하여 18 당량) 과 프로필렌글리콜 (2.921 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (44.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.87 mg/ml, 8.00 ml) was diluted with propylene glycol (4.00 ml). To this solution, a mixture of drug linker 17a dimethylsulfoxide solution (10 mM, 1.079 ml, 18 equivalents relative to 1 antibody molecule) and propylene glycol (2.921 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (44.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.16 ㎎/㎖ Antibody concentration: 1.16 mg/ml

항체 수량 : 51.5 ㎎ (59 ％) Antibody quantity: 51.5 mg (59%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 111 : 항체 약물 콘쥬게이트 26 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 3 의 합성) Example 111: Synthesis of Antibody Drug Conjugate 26 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 3)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.87 ㎎/㎖, 8.00 ㎖) 을 프로필렌글리콜 (4.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 17b 의 디메틸술폭시드 용액 (10 mM, 1.079 ㎖, 항체 1 분자에 대하여 18 당량) 과 프로필렌글리콜 (2.921 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (44.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.87 mg/ml, 8.00 ml) was diluted with propylene glycol (4.00 ml). To this solution, a mixture of drug linker 17b dimethylsulfoxide solution (10 mM, 1.079 ml, 18 equivalents relative to 1 antibody molecule) and propylene glycol (2.921 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (44.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.24 ㎎/㎖ Antibody concentration: 1.24 mg/ml

항체 수량 : 54.99 ㎎ (63 ％) Antibody quantity: 54.99 mg (63%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 112 : 항체 약물 콘쥬게이트 27 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 4 의 합성) Example 112: Synthesis of Antibody Drug Conjugate 27 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 4)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 19 의 디메틸술폭시드 용액 (10 mM, 0.180 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.320 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 19 dimethylsulfoxide solution (10 mM, 0.180 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.320 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.79 ㎎/㎖ Antibody concentration: 0.79 mg/ml

항체 수량 : 5.53 ㎎ (51 ％) Antibody quantity: 5.53 mg (51%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 113 : 항체 약물 콘쥬게이트 28 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 5 의 합성) Example 113: Synthesis of Antibody Drug Conjugate 28 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 5)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 21 의 디메틸술폭시드 용액 (10 mM, 0.180 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.320 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 21 dimethylsulfoxide solution (10 mM, 0.180 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.320 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.84 ㎎/㎖ Antibody concentration: 0.84 mg/ml

항체 수량 : 5.91 ㎎ (54 ％) Antibody quantity: 5.91 mg (54%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 114 : 항체 약물 콘쥬게이트 29 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 6 의 합성) Example 114: Synthesis of Antibody Drug Conjugate 29 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 6)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 22 의 디메틸술폭시드 용액 (10 mM, 0.180 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.320 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (5.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 22 dimethylsulfoxide solution (10 mM, 0.180 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.320 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (5.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.92 ㎎/㎖ Antibody concentration: 0.92 mg/ml

항체 수량 : 4.60 ㎎ (42 ％) Antibody quantity: 4.60 mg (42%)

약물 평균 결합수 : 3.5Average number of drug bonds: 3.5

실시예 115 : 항체 약물 콘쥬게이트 30 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 7 의 합성) Example 115: Synthesis of Antibody Drug Conjugate 30 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 7)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 18 의 디메틸술폭시드 용액 (10 mM, 0.180 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.320 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 18 dimethylsulfoxide solution (10 mM, 0.180 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.320 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.08 ㎎/㎖ Antibody concentration: 1.08 mg/ml

항체 수량 : 7.53 ㎎ (69 ％) Antibody quantity: 7.53 mg (69%)

약물 평균 결합수 : 3.9Average number of drug bonds: 3.9

실시예 116 : 항체 약물 콘쥬게이트 31 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 8 의 합성) Example 116: Synthesis of Antibody Drug Conjugate 31 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 8)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 20 의 디메틸술폭시드 용액 (10 mM, 0.180 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.320 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 20 dimethylsulfoxide solution (10 mM, 0.180 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.320 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.09 ㎎/㎖ Antibody concentration: 1.09 mg/ml

항체 수량 : 7.62 ㎎ (70 ％) Antibody quantity: 7.62 mg (70%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 117 : 항체 약물 콘쥬게이트 32 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 9 의 합성) Example 117: Synthesis of Antibody Drug Conjugate 32 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 9)

당사슬 리모델링 항체 8 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.48 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 17a 의 디메틸술폭시드 용액 (10 mM, 0.087 ㎖, 항체 1 분자에 대하여 12 당량) 과 프로필렌글리콜 (0.413 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 8 in phosphate buffered saline (pH 6.0) (10.48 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 17a dimethylsulfoxide solution (10 mM, 0.087 ml, 12 equivalents per antibody molecule) and propylene glycol (0.413 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.77 ㎎/㎖ Antibody concentration: 0.77 mg/ml

항체 수량 : 5.36 ㎎ (51 ％) Antibody quantity: 5.36 mg (51%)

약물 평균 결합수 : 1.8Average number of drug bonds: 1.8

실시예 118 : 항체 약물 콘쥬게이트 33 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 10 의 합성) Example 118: Synthesis of Antibody Drug Conjugate 33 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 10)

당사슬 리모델링 항체 8 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.48 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 17b 의 디메틸술폭시드 용액 (10 mM, 0.087 ㎖, 항체 1 분자에 대하여 12 당량) 과 프로필렌글리콜 (0.413 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 8 in phosphate buffered saline (pH 6.0) (10.48 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 17b dimethylsulfoxide solution (10 mM, 0.087 ml, 12 equivalents per antibody molecule) and propylene glycol (0.413 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.91 ㎎/㎖ Antibody concentration: 0.91 mg/ml

항체 수량 : 6.34 ㎎ (61 ％) Antibody quantity: 6.34 mg (61%)

약물 평균 결합수 : 1.8Average number of drug bonds: 1.8

실시예 119 : 항체 약물 콘쥬게이트 34 의 합성 (항HER2 항체 - CDN 콘쥬게이트 19 의 합성) Example 119: Synthesis of Antibody Drug Conjugate 34 (Synthesis of Anti-HER2 Antibody-CDN Conjugate 19)

참고예 1 에 따라서 조제한 항HER2 항체의 인산 완충 용액을, 공통 조작 C 에 따라서 버퍼 교환하고, 인산 완충 생리 식염수/5 mM - EDTA 의 항체 용액 (7.69 ㎎/㎖) 을 조제하였다. 이 항체 용액 (0.65 ㎖) 에 트리스(2-카르복시에틸)포스핀염산염의 수용액 (10 mM, 20.7 ㎕) 과 인산수소2칼륨 수용액 (1 M, 9.8 ㎕) 을 첨가하였다. 반응액의 pH 가 7.4 ± 1.0 이내인 것을 확인한 후, 37 ℃ 에서 2 시간 교반하였다. 약물 링커 23 의 DMSO 용액 (10 mM, 0.0689 ㎖) 을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 1 시간 반응시켰다. 반응액에 N-아세틸-L-시스테인 수용액 (100 mM, 6.9 ㎕) 을 첨가하여 반응을 정지시켰다. 반응액을 공통 조작 C 에 기재된 방법에 따라서 버퍼 교환하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (1.6 ㎖) 을 얻었다. The anti-HER2 antibody phosphate buffer solution prepared according to Reference Example 1 was buffer-exchanged according to common operation C to prepare an antibody solution (7.69 mg/ml) of phosphate buffered physiological saline/5 mM -EDTA. To this antibody solution (0.65 ml), an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (10 mM, 20.7 µl) and an aqueous solution of dipotassium hydrogen phosphate (1 M, 9.8 µl) were added. After confirming that the pH of the reaction solution was within 7.4±1.0, the mixture was stirred at 37°C for 2 hours. A DMSO solution (10 mM, 0.0689 ml) of drug linker 23 was added and reacted at room temperature for 1 hour using a tube rotator (MTR-103, As One Co., Ltd.). The reaction was stopped by adding an aqueous solution of N-acetyl-L-cysteine (100 mM, 6.9 µl) to the reaction solution. The reaction solution was buffer-exchanged according to the method described in Common Operation C to obtain an ABS solution (1.6 ml) of the antibody-drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.46 ㎎/㎖ Antibody concentration: 1.46 mg/ml

항체 수량 : 2.34 ㎎ (47 ％) Antibody quantity: 2.34 mg (47%)

약물 평균 결합수 : 6.5Average number of drug bonds: 6.5

실시예 120 : 약물 링커 24 의 합성 Example 120: Synthesis of Drug Linker 24

[합성 스킴][Synthesis Scheme]

[화학식 247][Formula 247]

Figure pat00620
Figure pat00620

(공정 1) (Process 1)

실시예 44 공정 8 에서 얻어진 화합물 (1.55 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 77 공정 6 에서 얻어진 화합물 (1.96 g) 을 사용하여, 실시예 1 공정 8 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.55 g) obtained in Example 44 Step 8, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O -[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[ An acetonitrile solution of cd]azulene was obtained. Using the obtained acetonitrile solution and the compound (1.96 g) obtained in Example 77 Step 6, a reaction was carried out in the same manner as in Example 1 Step 8, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-(트리메틸실릴)에틸 (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-하이드록시-2-옥소-10-술파닐리덴옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]아미노}-2-옥소에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cya noethoxy)-15-fluoro-2-hydroxy-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3, 2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl }ethoxy)methyl]amino}-2-oxoethyl)carbamate

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 62 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.07 g : 불순물 함유) 을 인 원자 상의 디아스테레오머 혼합물로서 얻었다. Using the crude product obtained in step 1 above, reaction was carried out in the same manner as in step 2 of Example 62 to obtain the title compound (1.07 g: containing impurities) as a mixture of diastereomers on a phosphorus atom.

MS(ESI)m/z : 1262(M＋H)＋.MS(ESI) m/z : 1262(M+H) + .

(공정 3) (Process 3)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-7-[6-옥소-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)-1,6-디하이드로-9H-푸린-9-일]-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2, 10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6 -dihydro-9H-purin-9-yl]-10-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene -2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10] Pentaoxadiphosphacyclotetradecin-2-oleate

상기 공정 2 에서 얻어진 (1.07 g) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 (1 : 1), 아세토니트릴 - 메탄올 (1 : 1) : 25 ％ - 90 ％ (0 분 - 40 분)] 로 정제하고, 표제 화합물의 디아스테레오머 1 (67.8 ㎎ : 불순물 함유) 과 디아스테레오머 2 (56.6 ㎎ : 불순물 함유) 를 얻었다. Using (1.07 g) obtained in the above step 2, the reaction was carried out in the same manner as in step 10 of Example 1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution/acetonitrile-methanol (1:1), acetonitrile-methanol (1:1): 25% - 90% (0 min - 40 min)] to obtain a diastereomer of the title compound 1 (67.8 mg: containing impurities) and diastereomer 2 (56.6 mg: containing impurities) were obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1105(M＋H)＋. MS(ESI) m/z : 1105(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1105(M＋H)＋.MS(ESI) m/z : 1105(M+H) + .

(공정 4-1) (Step 4-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,16R)-15-fluoro-7-(1-{2-[(glycylamino)methoxy] Ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dioxo-10-sulfide-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2-oleate

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 1) (67.8 ㎎) 을 사용하여, 실시예 11 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 (1 : 1), 아세토니트릴 - 메탄올 (1 : 1) : 10 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (33.7 ㎎ : 불순물 함유) 을 얻었다. Using the compound (diastereomer 1) (67.8 mg) obtained in step 3 above, reaction was carried out in the same manner as in Example 11 step 9-1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate] /acetonitrile] and by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile-methanol (1:1), acetonitrile-methanol (1:1): 10% - 60% (0 min - 30 min)] Purification gave the title compound (33.7 mg: containing impurities).

MS(ESI)m/z : 847(M＋H)＋.MS(ESI) m/z : 847(M+H) + .

(공정 4-2) (Process 4-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2-올레이트 Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,16R)-15-fluoro-7-(1-{2-[(glycylamino)methoxy] Ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dioxo-10-sulfide-14-(6,7,8,9 -Tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2-oleate

상기 공정 3 에서 얻어진 화합물 (디아스테레오머 2) (56.6 ㎎) 을 사용하여, 실시예 11 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 (1 : 1), 아세토니트릴 - 메탄올 (1 : 1) : 10 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (32.6 ㎎ : 불순물 함유) 을 얻었다. Using the compound (diastereomer 2) (56.6 mg) obtained in step 3 above, reaction was carried out in the same manner as in Example 11 step 9-1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate] /acetonitrile] and by preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile-methanol (1:1), acetonitrile-methanol (1:1): 10% - 60% (0 min - 30 min)] Purification gave the title compound (32.6 mg: containing impurities).

MS(ESI)m/z : 847(M＋H)＋.MS(ESI) m/z : 847(M+H) + .

(공정 5-1) (Process 5-1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2-옥사이드-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2-oxide -2,10-dioxo-10-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(디아스테레오머 1) (Diastereomer 1)

상기 공정 4-1 에서 얻어진 화합물 (33.7 ㎎) 과 실시예 22 공정 11 에서 얻어진 화합물 (21.3 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (11.2 ㎎) 을 얻었다. After reacting in the same manner as in Example 22 Step 9-1 using the compound (33.7 mg) obtained in Step 4-1 and the compound (21.3 mg) obtained in Step 11 of Example 22, the following [purification conditions ] to obtain the title compound (11.2 mg).

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 40 ％ - 90 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 40% - 90% (0 min - 40 min) ].

Figure pat00621
Figure pat00621

(공정 5-2) (Process 5-2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2-옥사이드-2,10-디옥소-10-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2-oxide -2,10-dioxo-10-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(디아스테레오머 2) (Diastereomer 2)

상기 공정 4-2 에서 얻어진 화합물 (32.6 ㎎) 과 실시예 22 공정 11 에서 얻어진 화합물 (20.6 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (16.7 ㎎) 을 얻었다. Using the compound (32.6 mg) obtained in the above step 4-2 and the compound (20.6 mg) obtained in Example 22 step 11, a reaction was conducted in the same manner as in Example 22 step 9-1, and then the following [purification conditions ] to obtain the title compound (16.7 mg).

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴], 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/메탄올, 메탄올 : 40 ％ - 90 ％ (0 분 - 40 분)], 및 분취 HPLC [100 mM 헥사플루오로-2-프로판올, 8 mM 트리에틸아민 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate/acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate/methanol, methanol: 40% - 90% (0 min - 40 min) ], and preparative HPLC [100 mM hexafluoro-2-propanol, 8 mM aqueous triethylamine/acetonitrile, acetonitrile: 10% - 45% (0 min - 40 min)].

Figure pat00622
Figure pat00622

실시예 121 : 약물 링커 25 의 합성 Example 121: Synthesis of Drug Linker 25

[합성 스킴][Synthesis Scheme]

[화학식 248][Formula 248]

Figure pat00623
Figure pat00623

(공정 1) (Process 1)

N,N-디에틸에탄아미늄 6-벤조일-2-{5-O-[비스(4-메톡시페닐)(페닐)메틸]-2-데옥시-2-플루오로-3-O-[옥사이드(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 N,N-diethylethanelaminium 6-benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-3-O-[ Oxide (oxo)-λ 5 -phosphanyl] -β-D-ribofuranosyl} -6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]a Julen

실시예 44 공정 7 에서 얻어진 화합물 (1.49 g) 의 피리딘 (10.4 ㎖) 용액에, 빙랭하에서 아인산디페닐 (599 ㎕) 을 첨가하고, 실온까지 승온하여 30 분간 교반하였다. 아인산디페닐 (200 ㎕) 을 추가하여, 2 시간 더 교반하였다. 빙랭하, 반응액에 물 (1.5 ㎖) 을 첨가하고, 실온에서 30 분간 교반한 후, 감압 농축하였다. 잔류물을 실리카 겔 칼럼 크로마토그래피 [아세트산에틸/메탄올/0.1 ％ 트리에틸아민] 으로 정제하고, 표제 화합물 (1.41 g) 을 얻었다. Example 44 Diphenyl phosphite (599 µl) was added to a solution of the compound (1.49 g) obtained in step 7 in pyridine (10.4 ml) under ice-cooling, and the mixture was heated to room temperature and stirred for 30 minutes. Diphenyl phosphite (200 µl) was added, and the mixture was stirred for another 2 hours. Under ice-cooling, water (1.5 ml) was added to the reaction solution, and after stirring at room temperature for 30 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate/methanol/0.1% triethylamine] to obtain the title compound (1.41 g).

Figure pat00624
Figure pat00624

(공정 2) (Process 2)

6-벤조일-2-{2-데옥시-2-플루오로-3-O-[하이드록시(옥소)-λ5-포스파닐]-β-D-리보퓨라노실}-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

상기 공정 1 에서 얻어진 화합물 (1.41 g) 의 디클로로메탄 (20.0 ㎖) 용액에, 물 (289 ㎕) 과 디클로로아세트산 (1.14 ㎖) 의 디클로로메탄 (20.0 ㎖) 용액을 첨가하고, 실온에서 10 분간 교반하였다. 반응액에 피리딘 (2.19 ㎖) 을 첨가하여 반응을 정지시킨 후, 감압 농축하였다. 잔류물에 트리플루오로아세트산피리딘염 (402 ㎎) 을 첨가하고, 탈수 아세토니트릴 (15 ㎖) 로 3 회 공비하고, 마지막 1 회는 10 ㎖ 정도의 아세토니트릴을 남겼다. 얻어진 아세토니트릴 용액을 그대로 다음의 반응에 사용하였다. To a dichloromethane (20.0 ml) solution of the compound (1.41 g) obtained in step 1 above, a dichloromethane (20.0 ml) solution of water (289 µl) and dichloroacetic acid (1.14 ml) was added, and the mixture was stirred at room temperature for 10 minutes. . After stopping the reaction by adding pyridine (2.19 ml) to the reaction solution, the mixture was concentrated under reduced pressure. Trifluoroacetic acid pyridine salt (402 mg) was added to the residue, and the mixture was azeotroped with dehydrated acetonitrile (15 ml) three times, leaving about 10 ml of acetonitrile at the last time. The obtained acetonitrile solution was used as it was for the next reaction.

MS(ESI)m/z : 477(M＋H)＋.MS(ESI) m/z : 477(M+H) + .

(공정 3) (Process 3)

실시예 77 공정 6 에서 얻어진 화합물 (2.15 g) 과 상기 공정 2 에서 얻어진 화합물의 아세토니트릴 용액을 사용하여, 실시예 63 공정 1 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Example 77 Using the compound (2.15 g) obtained in Step 6 and an acetonitrile solution of the compound obtained in Step 2, a reaction was carried out in the same manner as in Example 63 Step 1, and the obtained crude product was reacted as is in the following reaction. used in

(공정 4) (Process 4)

2-(트리메틸실릴)에틸 (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2,10-디옥소-2-술파닐옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]아미노}-2-옥소에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cya noethoxy)-15-fluoro-2,10-dioxo-2-sulfanyloctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy )methyl]amino}-2-oxoethyl)carbamate

상기 공정 3 에서 얻어진 미정제 생성물을 사용하여, 실시예 1 공정 9 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (1.00 g : 불순물 함유) 을 얻었다.The reaction was carried out in the same manner as in Example 1 Step 9 using the crude product obtained in Step 3 to obtain the title compound (1.00 g: containing impurities).

MS(ESI)m/z : 1262(M＋H)＋.MS(ESI) m/z : 1262(M+H) + .

(공정 5) (Process 5)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-부틸(디메틸)실릴]옥시}-15-플루오로-2,10-디옥소-7-[6-옥소-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)-1,6-디하이드로-9H-푸린-9-일]-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-올레이트 Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro- 2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1 ,6-dihydro-9H-purin-9-yl] -2-sulfide-14- (6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo [cd] Azulen-2-yl) octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecyn-10-oleate

상기 공정 4 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시하고, 표제 화합물의 디아스테레오머 1 (191 ㎎ : 불순물 함유) 과 디아스테레오머 2 (369 ㎎ : 불순물 함유) 를 얻었다. Using the compound (1.00 g) obtained in step 4 above, a reaction was carried out in the same manner as in step 10 of Example 1 to obtain diastereomer 1 (191 mg: containing impurities) and diastereomer 2 (369 mg) of the title compound. : containing impurities) was obtained.

디아스테레오머 1 (저극성) Diastereomer 1 (low polarity)

MS(ESI)m/z : 1105(M＋H)＋. MS(ESI) m/z : 1105(M+H) + .

디아스테레오머 2 (고극성) Diastereomer 2 (high polarity)

MS(ESI)m/z : 1105(M＋H)＋.MS(ESI) m/z : 1105(M+H) + .

(공정 6-1) (Step 6-1)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-올레이트 Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methyl Toxy] ethyl} -6-oxo-1,6-dihydro-9H-purin-9-yl) -16-hydroxy-2,10-dioxo-2-sulfide-14- (6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-oleate

상기 공정 5 에서 얻어진 화합물 (디아스테레오머 1) (191 ㎎) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (21.9 ㎎ : 불순물 함유) 을 얻었다. The reaction was carried out in the same manner as in Example 40 Step 5 using the compound (Diastereomer 1) (191 mg) obtained in Step 5 above to obtain the title compound (21.9 mg: containing impurities).

Figure pat00625
Figure pat00625

(공정 6-2) (Step 6-2)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-10-올레이트 Bis(N,N-diethylethanenaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methyl Toxy] ethyl} -6-oxo-1,6-dihydro-9H-purin-9-yl) -16-hydroxy-2,10-dioxo-2-sulfide-14- (6,7,8 ,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-oleate

상기 공정 5 에서 얻어진 화합물 (디아스테레오머 2) (369 ㎎) 을 사용하여, 실시예 40 공정 5 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (137 ㎎ : 불순물 함유) 을 얻었다. A reaction was carried out in the same manner as in Example 40 Step 5 using the compound (Diastereomer 2) (369 mg) obtained in Step 5 above to obtain the title compound (137 mg: containing impurities).

Figure pat00626
Figure pat00626

(공정 7-1) (Process 7-1)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-10-옥사이드-2,10-디옥소-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-10-oxide -2,10-dioxo-2-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(약물 링커 25a : 디아스테레오머 1) (Drug linker 25a: diastereomer 1)

상기 공정 6-1 에서 얻어진 화합물 (21.9 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물 (26.3 ㎎) 을 얻었다. The compound (21.9 mg) obtained in the above step 6-1 was reacted in the same manner as in Example 22 step 9-1, and then purified under the following [purification conditions] to obtain the title compound (26.3 mg). .

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 50 ％ (0 분 - 30 분)] 및 Sep-Pak (등록상표) C18 [물/아세토니트릴/0.1 ％ 트리에틸아민]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 50% (0 min - 30 min)] and Sep-Pak (registered trademark) C18 [water/acetonitrile/0.1 % triethylamine].

Figure pat00627
Figure pat00627

(공정 7-2) (Process 7-2)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-10-옥사이드-2,10-디옥소-2-술파이드-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-10-oxide -2,10-dioxo-2-sulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo tetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

(약물 링커 25b : 디아스테레오머 2) (Drug linker 25b: diastereomer 2)

상기 공정 6-2 에서 얻어진 화합물 (47.5 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제하고, 표제 화합물 (15.1 ㎎) 을 얻었다. The compound (47.5 mg) obtained in the above step 6-2 was reacted in the same manner as in Example 22 step 9-1, and then purified under the following [purification conditions] to obtain the title compound (15.1 mg). .

[정제 조건] 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴, 아세토니트릴 : 30 ％ - 50 ％ (0 분 - 30 분)] 및 Sep-Pak (등록상표) C18 [물/아세토니트릴/0.1 ％ 트리에틸아민]. [Purification conditions] Preparative HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile, acetonitrile: 30% - 50% (0 min - 30 min)] and Sep-Pak (registered trademark) C18 [water/acetonitrile/0.1 % triethylamine].

Figure pat00628
Figure pat00628

실시예 122 : 약물 링커 26 의 합성 Example 122: Synthesis of Drug Linker 26

[합성 스킴][Synthesis Scheme]

[화학식 249][Formula 249]

Figure pat00629
Figure pat00629

(공정 1) (Process 1)

실시예 77 공정 6 에서 얻어진 화합물 (1.26 g) 을 사용하여, 실시예 1 공정 7 과 동일한 수법으로 반응을 실시하고, 3'-O-[tert-부틸(디메틸)실릴]-2'-O-[하이드록시(옥소)-λ5-포스파닐]-1-(2-{[(N-{[2-(트리메틸실릴)에톡시]카르보닐}글리실)아미노]메톡시}에틸)이노신의 아세토니트릴 용액을 얻었다. 얻어진 아세토니트릴 용액과 실시예 44 공정 8 에서 얻어진 화합물 (1.00 g) 을 사용하여, 실시예 63 공정 1 과 동일한 수법으로 반응을 실시하고, 얻어진 미정제 생성물을 그대로 다음의 반응에 사용하였다.Using the compound (1.26 g) obtained in Example 77 Step 6, reaction was carried out in the same manner as in Example 1 Step 7 to obtain 3'-O-[tert-butyl(dimethyl)silyl]-2'-O- [hydroxy(oxo)-λ 5 -phosphanyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine An acetonitrile solution was obtained. Using the obtained acetonitrile solution and the compound (1.00 g) obtained in Example 44 Step 8, a reaction was carried out in the same manner as in Example 63 Step 1, and the obtained crude product was used as it was for the next reaction.

(공정 2) (Process 2)

2-(트리메틸실릴)에틸 (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]아미노}-2-옥소에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cya noethoxy)-15-fluoro-2-hydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy )methyl]amino}-2-oxoethyl)carbamate

상기 공정 1 에서 얻어진 미정제 생성물을 사용하여, 실시예 62 공정 2 와 동일한 수법으로 반응을 실시하고, 표제 화합물 (678 ㎎ : 불순물 함유) 을 얻었다. The reaction was carried out in the same manner as in Example 62 Step 2 using the crude product obtained in Step 1 above to obtain the title compound (678 mg: containing impurities).

MS(ESI)m/z : 1246(M＋H)＋.MS(ESI) m/z : 1246(M+H) + .

(공정 3) (Process 3)

2-(트리메틸실릴)에틸 (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-벤조일-6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)-16-{[tert-부틸(디메틸)실릴]옥시}-10-(2-시아노에톡시)-15-플루오로-2-하이드록시-2,10-디옥소옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]아미노}-2-옥소에틸)카르바메이트 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8, 9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cya noethoxy)-15-fluoro-2-hydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l ][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy )methyl]amino}-2-oxoethyl)carbamate

상기 공정 2 에서 얻어진 화합물 (678 ㎎) 을 사용하여, 실시예 1 공정 10 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 25 ％ - 90 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (99.6 ㎎ : 불순물 함유) 을 얻었다. Using the compound (678 mg) obtained in step 2 above, reaction was carried out in the same manner as in step 10 of Example 1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] and preparative HPLC [ 10 mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 25% - 90% (0 min - 30 min)] to obtain the title compound ( 99.6 mg: containing impurities) was obtained.

MS(ESI)m/z : 1089(M＋H)＋.MS(ESI) m/z : 1089(M+H) + .

(공정 4) (Process 4)

비스(N,N-디에틸에탄아미늄) (5R,7R,8R,12aR,14R,15R,16R)-15-플루오로-7-(1-{2-[(글리실아미노)메톡시]에틸}-6-옥소-1,6-디하이드로-9H-푸린-9-일)-16-하이드록시-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-2,10-비스(올레이트) Bis(N,N-diethylethanelaminium) (5R,7R,8R,12aR,14R,15R,16R)-15-fluoro-7-(1-{2-[(glycylamino)methoxy] Ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H -2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-2,10-bis(oleate)

상기 공정 3 에서 얻어진 화합물 (99.6 ㎎) 을 사용하여, 실시예 11 공정 9-1 과 동일한 수법으로 반응을 실시한 후, C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴 - 메탄올 용액 (1 : 1), 아세토니트릴 - 메탄올 용액 (1 : 1) : 10 ％ - 60 ％ (0 분 - 30 분)] 로 정제하고, 표제 화합물 (60.8 ㎎ : 불순물 함유) 을 얻었다. Using the compound (99.6 mg) obtained in step 3 above, the reaction was carried out in the same manner as in Example 11 step 9-1, followed by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] and fractionation Purified by HPLC [10 mM aqueous solution of triethylammonium acetate/acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10% - 60% (0 min - 30 min)], A compound (60.8 mg: containing impurities) was obtained.

MS(ESI)m/z : 831(M＋H)＋.MS(ESI) m/z : 831(M+H) + .

(공정 5) (Process 5)

비스(N,N-디에틸에탄아미늄) N-[4-(11,12-디데히드로디벤조[b,f]아조신-5(6H)-일)-4-옥소부타노일]글리실글리실-L-페닐알라닐-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-플루오로-16-하이드록시-2,10-디옥사이드-2,10-디옥소-14-(6,7,8,9-테트라하이드로-2H-2,3,5,6-테트라아자벤조[cd]아줄렌-2-일)옥타하이드로-2H,10H,12H-5,8-메타노-2λ5,10λ5-푸로[3,2-l][1,3,6,9,11,2,10]펜타옥사디포스파시클로테트라데신-7-일]-6-옥소-6,9-디하이드로-1H-푸린-1-일}에톡시)메틸]글리신아미드 Bis(N,N-diethylethanenaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycyl Glycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10 -Dioxide-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro- 2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide

상기 공정 4 에서 얻어진 화합물 (60.8 ㎎) 과 실시예 22 공정 11 에서 얻어진 화합물 (21.3 ㎎) 을 사용하여, 실시예 22 공정 9-1 과 동일한 수법으로 반응을 실시한 후, 이하의 [정제 조건] 으로 정제를 실시하고, 표제 화합물 (30.5 ㎎) 을 얻었다. After reacting in the same manner as in Example 22 Step 9-1 using the compound (60.8 mg) obtained in Step 4 and the compound (21.3 mg) obtained in Step 11 of Example 22, the following [Purification conditions] Purification was carried out to obtain the title compound (30.5 mg).

[정제 조건] C18 실리카 겔 칼럼 크로마토그래피 [10 mM 아세트산트리에틸암모늄 수용액/아세토니트릴] 과 분취 HPLC [100 mM 헥사플루오로-2-프로판올, 8 mM 트리에틸아민 수용액/아세토니트릴, 아세토니트릴 : 10 ％ - 45 ％ (0 분 - 40 분)]. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate/acetonitrile] and preparative HPLC [100 mM hexafluoro-2-propanol, 8 mM aqueous solution of triethylamine/acetonitrile, acetonitrile: 10 % - 45% (0 min - 40 min)].

Figure pat00630
Figure pat00630

실시예 123 : 항체 약물 콘쥬게이트 35 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 11 의 합성) Example 123: Synthesis of Antibody Drug Conjugate 35 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 11)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.87 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 24a 의 디메틸술폭시드 용액 (10 mM, 0.135 ㎖, 항체 1 분자에 대하여 18 당량) 과 프로필렌글리콜 (0.365 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.87 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 24a dimethylsulfoxide solution (10 mM, 0.135 ml, 18 equivalents per antibody molecule) and propylene glycol (0.365 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 F 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and F, and the following results were obtained.

항체 농도 : 1.08 ㎎/㎖ Antibody concentration: 1.08 mg/ml

항체 수량 : 7.54 ㎎ (69 ％) Antibody quantity: 7.54 mg (69%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 124 : 항체 약물 콘쥬게이트 36 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 12 의 합성) Example 124: Synthesis of Antibody Drug Conjugate 36 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 12)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.87 ㎎/㎖, 1.00 ㎖) 을 프로필렌글리콜 (0.500 ㎖) 로 희석하였다. 이 용액에 약물 링커 24b 의 디메틸술폭시드 용액 (10 mM, 0.135 ㎖, 항체 1 분자에 대하여 18 당량) 과 프로필렌글리콜 (0.365 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (7.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.87 mg/ml, 1.00 ml) was diluted with propylene glycol (0.500 ml). To this solution, a mixture of drug linker 24b dimethylsulfoxide solution (10 mM, 0.135 ml, 18 equivalents relative to 1 antibody molecule) and propylene glycol (0.365 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (7.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 F 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and F, and the following results were obtained.

항체 농도 : 1.12 ㎎/㎖ Antibody concentration: 1.12 mg/ml

항체 수량 : 7.85 ㎎ (72 ％) Antibody quantity: 7.85 mg (72%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 125 : 항체 약물 콘쥬게이트 37 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 13 의 합성) Example 125: Synthesis of Antibody Drug Conjugate 37 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 13)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.87 ㎎/㎖, 2.00 ㎖) 을 프로필렌글리콜 (1.00 ㎖) 로 희석하였다. 이 용액에 약물 링커 26 의 디메틸술폭시드 용액 (10 mM, 0.270 ㎖, 항체 1 분자에 대하여 18 당량) 과 프로필렌글리콜 (0.730 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (14.0 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.87 mg/ml, 2.00 ml) was diluted with propylene glycol (1.00 ml). To this solution, a mixture of drug linker 26 dimethylsulfoxide solution (10 mM, 0.270 ml, 18 equivalents per antibody molecule) and propylene glycol (0.730 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (14.0 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 F 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and F, and the following results were obtained.

항체 농도 : 1.05 ㎎/㎖ Antibody concentration: 1.05 mg/ml

항체 수량 : 14.71 ㎎ (68 ％) Antibody quantity: 14.71 mg (68%)

약물 평균 결합수 : 3.8Average number of drug bonds: 3.8

실시예 126 : 항체 약물 콘쥬게이트 38 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 14 의 합성) Example 126: Synthesis of Antibody Drug Conjugate 38 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 14)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 3.00 ㎖) 을 프로필렌글리콜 (1.50 ㎖) 로 희석하였다. 이 용액에 약물 링커 25a 의 디메틸술폭시드 용액 (10 mM, 0.540 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.960 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 3 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (19 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 3.00 ml) was diluted with propylene glycol (1.50 ml). To this solution, a mixture of drug linker 25a dimethylsulfoxide solution (10 mM, 0.540 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.960 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 3 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (19 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 1.38 ㎎/㎖ Antibody concentration: 1.38 mg/ml

항체 수량 : 26.28 ㎎ (80 ％) Antibody quantity: 26.28 mg (80%)

약물 평균 결합수 : 3.7Average number of drug bonds: 3.7

실시예 127 : 항체 약물 콘쥬게이트 39 의 합성 (항HER2 항체 2 - CDN 콘쥬게이트 15 의 합성) Example 127: Synthesis of Antibody Drug Conjugate 39 (Synthesis of Anti-HER2 Antibody 2 - CDN Conjugate 15)

당사슬 리모델링 항체 4 의 인산 완충 생리 식염수 (pH 6.0) 용액 (10.89 ㎎/㎖, 0.500 ㎖) 을 프로필렌글리콜 (0.250 ㎖) 로 희석하였다. 이 용액에 약물 링커 25b 의 디메틸술폭시드 용액 (10 mM, 0.090 ㎖, 항체 1 분자에 대하여 24 당량) 과 프로필렌글리콜 (0.160 ㎖) 의 혼합액을 첨가하고, 튜브 로테이터 (MTR-103, 애즈원 주식회사) 를 사용하여 실온에서 2 일간 반응시켰다. 반응액을 공통 조작 D 에 기재된 방법에 따라서 정제하고, 목적으로 하는 항체 약물 콘쥬게이트의 ABS 용액 (3.5 ㎖) 을 얻었다. A solution of sugar chain remodeling antibody 4 in phosphate buffered saline (pH 6.0) (10.89 mg/ml, 0.500 ml) was diluted with propylene glycol (0.250 ml). To this solution, a mixture of drug linker 25b dimethylsulfoxide solution (10 mM, 0.090 ml, 24 equivalents relative to 1 antibody molecule) and propylene glycol (0.160 ml) was added, and a tube rotator (MTR-103, As One Co., Ltd.) was added. and reacted at room temperature for 2 days. The reaction solution was purified according to the method described in Common Operation D to obtain an ABS solution (3.5 ml) of the antibody drug conjugate of interest.

공통 조작 E 및 G 에 기재된 방법에 따라서 분석하여, 하기의 결과를 얻었다. Analysis was performed according to the methods described in Common Operations E and G, and the following results were obtained.

항체 농도 : 0.93 ㎎/㎖ Antibody concentration: 0.93 mg/ml

항체 수량 : 3.25 ㎎ (60 ％) Antibody quantity: 3.25 mg (60%)

약물 평균 결합수 : 3.5Average number of drug bonds: 3.5

(참고예 1 : 항HER2 항체의 제조) (Reference Example 1: Preparation of anti-HER2 antibody)

본 명세서에 있어서, 「트라스투주맙」 은 HERCEPTIN (등록상표), huMAb4D5-8, rhuMAb4D5-8 이라고 불리는 경우도 있으며, 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 2 에 기재된 아미노산 서열로 이루어지는 중사슬을 갖는 인간화 IgG1 항체이다. 아미노산 서열은 US5821337 을 참조하였다. 트라스투주맙의 경사슬 아미노산 서열 (서열 번호 1) 및 중사슬 아미노산 서열 (서열 번호 2) 을 도 4 에 나타낸다.In the present specification, "trastuzumab" is sometimes called HERCEPTIN (registered trademark), huMAb4D5-8, or rhuMAb4D5-8, and has a light chain composed of the amino acid sequence shown in SEQ ID NO: 1 and an amino acid sequence shown in SEQ ID NO: 2. It is a humanized IgG1 antibody having a heavy chain consisting of See US5821337 for amino acid sequences. The light chain amino acid sequence (SEQ ID NO: 1) and heavy chain amino acid sequence (SEQ ID NO: 2) of Trastuzumab are shown in FIG. 4 .

본 명세서에서 사용한 항HER2 항체는, 트라스투주맙의 중사슬 아미노산 서열의 EU 인덱스 234 번째 및 235 번째의 류신 (L) 을 알라닌 (A) 으로 변이 (본 명세서 중, LALA 변이라고도 한다) 시킨, 트라스투주맙의 정상 영역 개변 IgG1 항체 (본 명세서 중, 개변 항HER2 항체라고도 한다) 를 설계하고, 제조하였다. 개변 항HER2 항체의 경사슬 아미노산 서열 (서열 번호 1) 및 중사슬 아미노산 서열 (서열 번호 3) 을 도 5 에 나타낸다.The anti-HER2 antibody used herein is a trastuzumab heavy chain amino acid sequence in which EU index 234th and 235th leucine (L) is mutated (in this specification, also referred to as LALA mutation) to alanine (A). A constant region modified IgG1 antibody of stuzumab (in the present specification, also referred to as a modified anti-HER2 antibody) was designed and produced. 5 shows the light chain amino acid sequence (SEQ ID NO: 1) and heavy chain amino acid sequence (SEQ ID NO: 3) of the modified anti-HER2 antibody.

(참고예 2 : 항LPS 항체의 제조) (Reference Example 2: Preparation of anti-LPS antibody)

항LPS 항체는 WO2015/046505 를 참조하여 제조하였다. 본 실시예에서 사용한 항LPS 항체는, 아이소타입이 IgG1 이며, LALA 변이를 갖는다 (본 명세서 중, 개변 항LPS 항체라고도 한다). 본 실시예에서 사용한 개변 항LPS 항체의 경사슬 및 중사슬의 아미노산 서열을 서열 번호 26 및 서열 번호 27 에 나타낸다.An anti-LPS antibody was prepared with reference to WO2015/046505. The anti-LPS antibody used in this Example has an isotype of IgG1 and has a LALA mutation (herein, it is also referred to as a modified anti-LPS antibody). SEQ ID NO: 26 and SEQ ID NO: 27 show the amino acid sequences of the light chain and heavy chain of the modified anti-LPS antibody used in this Example.

(참고예 3 : ML-RR-CDA·2Na＋ 의 합성) (Reference Example 3: Synthesis of ML-RR-CDA·2Na + )

본 명세서에서, 레퍼런스 화합물로서 사용한 ML-RR-CDA·2Na＋ 는, 특허문헌 3 (WO2014/189805) 에 기재된 방법에 따라서 합성하였다.In this specification, ML-RR-CDA·2Na + used as a reference compound was synthesized according to the method described in Patent Document 3 (WO2014/189805).

(참고예 4 : 2',3'-cGAMP 의 합성) (Reference Example 4: Synthesis of 2',3'-cGAMP)

본 명세서에서, 레퍼런스 화합물로서 사용한 2',3'-cGAMP 는, cGAS 를 사용하여 ATP 와 GTP 로부터 효소적으로 합성하였다. cGAS 의 조제 및 효소 반응은, 문헌 기재의 방법 (I㎜unity, 2013, 39, 1019-1031, Cell Rep. 2014, 6, 421-430) 을 적절히 개변하여 실시하였다. 정제는, 약염기성 음이온 교환 수지 (DIAION WA10) 및 합성 흡착제 (SEPABEADS SP207SS) 를 사용한 칼럼 크로마토그래피에 의해 실시하였다.In this specification, 2',3'-cGAMP used as a reference compound was enzymatically synthesized from ATP and GTP using cGAS. Preparation of cGAS and enzyme reaction were carried out by appropriately modifying the method described in the literature (Immunity, 2013, 39, 1019-1031, Cell Rep. 2014, 6, 421-430). Purification was carried out by column chromatography using a weak basic anion exchange resin (DIAION WA10) and a synthetic adsorbent (SEPABEADS SP207SS).

(참고예 5 : 항HER2 항체 2 의 제조) (Reference Example 5: Preparation of anti-HER2 antibody 2)

「페르투주맙」 은 PERJETA (등록상표) 라고 불리는 경우도 있으며, 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 29 에 기재된 아미노산 서열로 이루어지는 중사슬을 갖는 인간화 IgG1 항체이다. 아미노산 서열은 WO2004/008099 를 참조하였다. 본 명세서에서는 항HER2 항체 2 라고도 한다. 페르투주맙의 경사슬 아미노산 서열 (서열 번호 28) 및 중사슬 아미노산 서열 (서열 번호 29) 을 도 17 에 나타낸다."Pertuzumab", sometimes called PERJETA (registered trademark), is a humanized IgG1 antibody having a light chain composed of the amino acid sequence shown in SEQ ID NO: 28 and a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 29. For amino acid sequences, reference was made to WO2004/008099. Also referred to as anti-HER2 antibody 2 in the present specification. The light chain amino acid sequence (SEQ ID NO: 28) and heavy chain amino acid sequence (SEQ ID NO: 29) of Pertuzumab are shown in FIG. 17 .

본 명세서에서는, LALA 변이에 더하여, 중사슬 아미노산 서열의 EU 인덱스 214 번째의 리신 (K) 을 아르기닌 (R) 으로 변이시킨 G1m3 알로타입의 정상 영역을 갖는 항HER2 항체 2 를 설계하고, 제조하였다 (본 명세서 중, 개변 항HER2 항체 2라고도 한다). 본 실시예에서 사용한 개변 항HER2 항체 2 의 경사슬 아미노산 서열 (서열 번호 28) 및 중사슬 아미노산 서열 (서열 번호 30) 을 도 18 에 나타낸다.In the present specification, in addition to LALA mutation, anti-HER2 antibody 2 having a G1m3 allotype constant region in which lysine (K) at EU index 214 of the heavy chain amino acid sequence was mutated to arginine (R) was designed and manufactured ( In this specification, also referred to as modified anti-HER2 antibody 2). 18 shows the light chain amino acid sequence (SEQ ID NO: 28) and heavy chain amino acid sequence (SEQ ID NO: 30) of the modified anti-HER2 antibody 2 used in this Example.

(참고예 6 : 항CD33 항체의 제조) (Reference Example 6: Preparation of anti-CD33 antibody)

항CD33 항체는 WO2014/057687 을 참조하여 제조하였다. 본 실시예에서 사용한 항CD33 항체는, 아이소타입이 IgG1 이며, LALA 변이를 갖는다. 본 실시예에서 사용한 항CD33 항체의 경사슬 아미노산 서열 (서열 번호 31) 및 중사슬 아미노산 서열 (서열 번호 32) 을 도 19 에 나타낸다.Anti-CD33 antibody was prepared with reference to WO2014/057687. The anti-CD33 antibody used in this Example has an isotype of IgG1 and has a LALA mutation. 19 shows the light chain amino acid sequence (SEQ ID NO: 31) and heavy chain amino acid sequence (SEQ ID NO: 32) of the anti-CD33 antibody used in this Example.

(참고예 7 : 항EphA2 항체의 제조) (Reference Example 7: Preparation of anti-EphA2 antibody)

항EphA2 항체는 WO2009/028639 를 참조하여 제조하였다. 본 실시예에서 사용한 항EphA2 항체는, 아이소타입이 IgG1 이다. 본 실시예에서 사용한 항EphA2 항체의 경사슬 아미노산 서열 (서열 번호 33) 및 중사슬 아미노산 서열을 (서열 번호 34) 을 도 20 에 나타낸다.An anti-EphA2 antibody was prepared with reference to WO2009/028639. The anti-EphA2 antibody used in this Example has an isotype of IgG1. 20 shows the light chain amino acid sequence (SEQ ID NO: 33) and heavy chain amino acid sequence (SEQ ID NO: 34) of the anti-EphA2 antibody used in this Example.

(참고예 8 : 항CDH6 항체의 제조) (Reference Example 8: Preparation of anti-CDH6 antibody)

항CDH6 항체는 WO2018/212136 을 참조하여 제조하였다. 본 실시예에서 사용한 항CDH6 항체는, 아이소타입이 IgG1 이며, LALA 변이에 더하여, 중사슬 아미노산 서열의 EU 인덱스 329 번째의 프롤린 (P) 을 글리신 (G) 으로 한 변이를 갖는다. 본 실시예에서 사용한 항CDH6 항체의 경사슬 아미노산 서열 (서열 번호 35) 및 중사슬 아미노산 서열을 (서열 번호 36) 을 도 21 에 나타낸다.Anti-CDH6 antibody was prepared with reference to WO2018/212136. The anti-CDH6 antibody used in this example has an IgG1 isotype, and in addition to the LALA mutation, has a mutation in which proline (P) at EU index 329 of the heavy chain amino acid sequence is changed to glycine (G). 21 shows the light chain amino acid sequence (SEQ ID NO: 35) and heavy chain amino acid sequence (SEQ ID NO: 36) of the anti-CDH6 antibody used in this Example.

(시험예 1) 리포터 세포를 사용한 STING 아고니스트 활성 평가 (Test Example 1) Evaluation of STING agonist activity using reporter cells

＜리포터 유전자 어세이＞ <Reporter gene assay>

인간 STING 아고니스트 활성은, STING 경로의 하류에 있는 인터페론 제어 인자 3 (interferon regulatory factor-3 (IRF3)) 의 경로의 활성화를 확인할 수 있는 THP1-DualTM 세포 (HAQ 변이형) (InvivoGen, CA, US) 를 사용하여 평가하였다. 마우스 STING 아고니스트 활성은 RAW-DualTM 세포 (InvivoGen) 를 사용하여 평가하였다.Human STING agonist activity was determined by THP1-Dual TM cells (HAQ variant) (InvivoGen, CA, which can confirm activation of the interferon regulatory factor-3 (IRF3) pathway downstream of the STING pathway) US) was used. Mouse STING agonist activity was evaluated using RAW-Dual TM cells (InvivoGen).

어세이는 이하와 같이 실시하였다. 처음에, 투명 96 웰 플레이트 (Corning, NY, US) 에, PBS 로 희석한 피험 화합물을 20 ㎕/웰로 분주 (分注) 하였다. 이어서, 어세이 버퍼 (10 ％ 소 혈청 알부민을 포함하는 RPMI1640 배지 또는 DMEM 배지) 에 현탁한 리포터 세포를 180 ㎕/웰 (1 × 105 세포/웰) 로 첨가하고 자극을 개시하였다. 37 ℃, 5 ％ CO2 환경하에서 24 시간 배양한 후의 원심 상청을 회수하였다. 6 ㎕ 의 회수한 상청을 백색 384 웰 플레이트에 첨가하고, 거기에 QUANTI-Luc (InvivoGen) 용액을 15 ㎕ 첨가하였다. 잘 혼화한 후, 플레이트 리더 (PerkinElmer, MA, US) 를 사용하여 발광을 측정하였다. 1.37 내지 100 μM 의 ML-RR-CDA·2Na＋ (Compound 21 in WO2014/189805) 를 처리한 세포에 있어서의 최대 카운트값을 100 ％, PBS 로 처리한 세포에서의 카운트를 0 ％ 로 하고, 피험 화합물이 50 ％ 의 카운트를 얻는 데에 필요한 농도를 EC50 (μM) 값으로 하여 GraphPad Prism (GraphPad Software, CA, US) 을 사용하여 산출하였다. 표 1 에 인간 STING 아고니스트 활성 시험의 결과를 나타낸다.The assay was performed as follows. Initially, 20 μl/well of a test compound diluted with PBS was dispensed into a transparent 96-well plate (Corning, NY, US). Next, reporter cells suspended in assay buffer (RPMI1640 medium or DMEM medium containing 10% bovine serum albumin) were added at 180 μl/well (1×10 5 cells/well) and stimulation was initiated. After culturing for 24 hours in a 37°C, 5% CO 2 environment, the centrifugal supernatant was collected. 6 μl of the recovered supernatant was added to a white 384 well plate, and 15 μl of QUANTI-Luc (InvivoGen) solution was added thereto. After mixing well, luminescence was measured using a plate reader (PerkinElmer, MA, US). The maximum count value in cells treated with 1.37 to 100 μM of ML-RR-CDA 2Na + (Compound 21 in WO2014/189805) was set to 100% and the count in cells treated with PBS was set to 0%, and the test The concentration required for the compound to obtain a count of 50% was calculated using GraphPad Prism (GraphPad Software, CA, US) as the EC50 (μM) value. Table 1 shows the results of the human STING agonist activity test.

[표 1][Table 1]

Figure pat00631
Figure pat00631

Figure pat00632
Figure pat00632

Figure pat00633
Figure pat00633

이 결과, 본원 화합물이 인간 STING 에 대한 아고니스트 활성을 갖는 것이 분명해졌다. 또, 마우스 STING 에 대해서도, 기존의 CDN 과 동등 이상의 아고니스트 활성을 확인하였다.As a result, it became clear that the present compound has an agonist activity against human STING. Also, for mouse STING, agonist activity equal to or higher than that of existing CDNs was confirmed.

(시험예 2) 리콤비넌트 STING C말 결합 도메인 단백질을 사용한 Protein thermal shift 어세이(Test Example 2) Protein thermal shift assay using recombinant STING C-terminal binding domain protein

(i) 각종 발현 플라스미드의 구축(i) Construction of various expression plasmids

＜인간 TMEM173 발현 플라스미드 (Plasmid) 의 구축＞ <Construction of human TMEM173 expression plasmid>

인간 STING (본 명세서 중, 인간 TMEM173 이라고 칭하는 경우도 있다) 의 포유 동물 세포 발현용 플라스미드는, 232 번째의 아르기닌 (R) 이 히스티딘 (H) 으로 변이한 (본 명세서 중, H232 변이 또는 REF 변이라고 한다) 인간 TMEM173 cDNA Clone (Accession NM＿198282.3, H232 (REF) 변이형 STING 발현 플라스미드) (GeneCopoeia, MD, US) 을 구입하였다. 인간 H232 (REF) 변이형 STING 의 아미노산 서열을 서열 번호 4 에, 뉴클레오티드 서열을 서열 번호 5 에 나타낸다. 또, H232 변이형 STING 발현 플라스미드를 주형 (鑄型) 으로 하고, Inverse PCR 법에 기초하는 부위 특이적 변이 도입을 실시하여 야생형 STING 및 변이형 STING 의 발현 플라스미드를 제조하였다. 상세하게는, 먼저 2 종류의 프라이머 (5'-CGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R(WT)fwd) (서열 번호 12) 및 5'-GTCACCGGTCTGCTGGGGCAGTTTATC-3' (H232R(WT)rev)) (서열 번호 13) 와 KOD-Plus-Mutagenesis 키트 (SMK-101) (토요보) 를 사용하여 PCR 을 실시하고, DNA 시퀀싱에 의해 목적으로 하는 야생형 (R232) STING 발현 플라스미드의 구축을 확인하였다. 인간 야생형 STING 의 아미노산 서열을 서열 번호 6 에, 뉴클레오티드 서열을 서열 번호 7 에 나타낸다.In the plasmid for expressing human STING (sometimes referred to as human TMEM173 in this specification) in mammalian cells, the 232nd arginine (R) is mutated to histidine (H) (in this specification, it is referred to as H232 mutation or REF mutation ) Human TMEM173 cDNA Clone (Accession NM_198282.3, H232 (REF) mutant STING expression plasmid) (GeneCopoeia, MD, US) was purchased. The amino acid sequence of human H232 (REF) variant STING is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 5. In addition, using the H232 mutant STING expression plasmid as a template, site-directed mutagenesis based on the inverse PCR method was performed to prepare wild-type STING and mutant STING expression plasmids. Specifically, first two types of primers (5'-CGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R(WT)fwd) (SEQ ID NO: 12) and 5'-GTCACCGGTCTGCTGGGGCAGTTTATC-3' (H232R (WT) rev)) (SEQ ID NO: 13) and KOD-Plus-Mutagenesis kit (SMK-101) (Toyobo), PCR was performed, and construction of the wild-type (R232) STING expression plasmid of interest was confirmed by DNA sequencing. The amino acid sequence of human wild-type STING is shown in SEQ ID NO: 6, and the nucleotide sequence is shown in SEQ ID NO: 7.

다음으로, HAQ (R71H, G230A 및 R293Q) 변이형을, 야생형 STING 발현 플라스미드와 동일한 수법으로 제조하였다. 상세하게는, H232 변이형 STING 발현 플라스미드를 주형으로 하고, 2 종류의 프라이머 (5'-GCTGACCGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R/G230A fwd) (서열 번호 14) 및 5'-GGTCTGCTGGGGCAGTTTATCCAGG-3' (H232R/G230A rev) (서열 번호 15)) 와 Mutagenesis 키트를 사용하여 PCR 을 실시. 2 개 지점에 동시에 변이를 도입함으로써, G230A 변이형 STING 플라스미드를 취득하였다. 또한, G230A 변이형 STING 발현 플라스미드를 주형으로 하고, 2 종류의 프라이머 (5'-CACCACATCCACTCCAGGTACCGG-3' (R71H fwd) (서열 번호 16) 및 5'-CAGCTCCTCAGCCAGGCTGCAGAC-3' (R71H rev) (서열 번호 17)) 와 Mutagenesis 키트를 사용하여 PCR 을 실시하여, R71H/G230A 변이형 STING 발현 플라스미드를 취득하였다.Next, mutant forms of HAQ (R71H, G230A and R293Q) were prepared by the same method as the wild type STING expression plasmid. Specifically, using the H232 mutant STING expression plasmid as a template, two primers (5'-GCTGACCGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R/G230A fwd) (SEQ ID NO: 14) and 5'-GGTCTGCTGGGGCAGTTTATCCAGG-3' (H232R/G230A Rev) (SEQ ID NO: 15)) and PCR using the Mutagenesis kit. By introducing mutations at two sites simultaneously, a G230A mutant STING plasmid was obtained. In addition, using the G230A mutant STING expression plasmid as a template, two primers (5'-CACCACATCCACTCCAGGTACCGG-3' (R71H fwd) (SEQ ID NO: 16) and 5'-CAGCTCCTCAGCCAGGCTGCAGAC-3' (R71H rev) (SEQ ID NO: 17 )) and a Mutagenesis kit, PCR was performed to obtain an R71H/G230A mutant STING expression plasmid.

계속해서, R71H/G230A 변이형의 STING 발현 플라스미드를 주형으로 하고, 2 종류의 프라이머 (5'-CAGACACTTGAGGACATCCTGGCAG-3' (R293Q fwd) (서열 번호 18) 및 5'-GCAGAAGAGTTTGGCCTGCTCAA-3' (R293Q rev) (서열 번호 19)) 와 Mutagenesis 키트를 사용하여 PCR 을 실시하고, HAQ (R71H/G230A/R293Q) 변이형의 발현 플라스미드를 취득하였다. 인간 HAQ 변이형 STING 의 아미노산 서열을 서열 번호 8 에, 뉴클레오티드 서열을 서열 번호 9 에 나타낸다.Subsequently, using the R71H/G230A mutant STING expression plasmid as a template, two types of primers (5'-CAGACACTTGAGGACATCCTGGCAG-3' (R293Q fwd) (SEQ ID NO: 18) and 5'-GCAGAAGAGTTTGGCCTGCTCAA-3' (R293Q rev) (SEQ ID NO: 19)) and a Mutagenesis kit, PCR was performed to obtain a mutant expression plasmid of HAQ (R71H/G230A/R293Q). The amino acid sequence of human HAQ variant STING is shown in SEQ ID NO: 8 and the nucleotide sequence is shown in SEQ ID NO: 9.

인간의 야생형 STING, REF 형 STING, HAQ 형 STING 의 아미노산 서열을 도 6 에 나타낸다.The amino acid sequences of human wild-type STING, REF-type STING, and HAQ-type STING are shown in FIG. 6 .

＜리콤비넌트 STING C말 결합 도메인 단백질 발현 플라스미드 등의 구축＞ <Construction of recombinant STING C-terminal binding domain protein expression plasmid, etc.>

인간 STING C말 결합 도메인 (aa139-342) 단백질 (UniProt entry Q86WV6) cDNA 는, 전체 길이의 인간 TMEM173 cDNA clone 발현 플라스미드 (야생형, H232 변이형 및 HAQ 변이형) 로부터 2 종류의 프라이머 (5'-ACCTGTATTTTCAGGGCCTGGCCCCAGCTGAGATCTCTG-3' (hST Fw＿v2) (서열 번호 20) 및 5'-CAGAATTCGCAAGCTTTTAAGTAACCTCTTCCTTTTCCTCCTGC-3' (hST Rv＿V3) (서열 번호 21)) 를 사용한 PCR 로 제조하였다. PCR 산물은, N말에 히스티딘 6 염기로 이루어지는 6xHis 태그, Avidin 태그 및 TEV 프로테아제 절단 사이트를 갖도록, In-Fusion HD Cloning 키트 (다카라 바이오) 를 사용하여 대장균 발현용 벡터인 pET15b 에 삽입하고, pET15b-HisAviTEV-hSTING (139-342) 인간 야생형, pET15b-HisAviTEV-hSTING (139-342) 인간 REF 변이형 및 pET15b-HisAviTEV-hSTING (139-342) 인간 AQ 변이형의 발현 플라스미드를 구축하였다.The human STING C-terminal binding domain (aa139-342) protein (UniProt entry Q86WV6) cDNA was prepared from the full-length human TMEM173 cDNA clone expression plasmid (wild type, H232 mutant and HAQ mutant) using two primers (5'-ACCTGTATTTTCAGGGCCTGGCCCCAGCTGAGATCTCTG -3' (hST Fw_v2) (SEQ ID NO: 20) and 5'-CAGAATTCGCAAGCTTTTAAGTAACCTCTTCCTTTTCCTCCTGC-3' (hST Rv_V3) (SEQ ID NO: 21)). The PCR product was inserted into pET15b, an E. coli expression vector, using an In-Fusion HD Cloning kit (Takara Bio) to have a 6xHis tag consisting of 6 bases of histidine at the N terminus, an Avidin tag, and a TEV protease cleavage site, and pET15b- Expression plasmids of HisAviTEV-hSTING (139-342) human wild type, pET15b-HisAviTEV-hSTING (139-342) human REF variant and pET15b-HisAviTEV-hSTING (139-342) human AQ variant were constructed.

마우스 STING C말 결합 도메인 (aa138-341) 단백질 (UniProt entry Q3TBT3) 발현용 cDNA 에는, 마우스 TMEM173 cDNA 서열로부터 138 번째 내지 341 번째의 아미노산에 해당하는 cDNA 를 eurofins Genomics 로 인공 합성한 것을 사용하였다. 마우스 STING 의 아미노산 서열을 서열 번호 10 에, 뉴클레오티드 서열을 서열 번호 11 에 나타낸다. 합성한 cDNA 는, N말에 히스티딘 6 염기로 이루어지는 6xHis 태그, Avidin 태그 및 TEV 프로테아제 절단 사이트를 갖도록 In-Fusion HD Cloning 키트를 사용하여 대장균 발현용 벡터인 pET15b 에 삽입하고, pET15b-HisAviTEV-mSTING (138-341) 마우스 야생형의 발현 플라스미드를 구축하였다.As cDNA for expressing the mouse STING C-terminal binding domain (aa138-341) protein (UniProt entry Q3TBT3), a cDNA corresponding to amino acids 138 to 341 from the mouse TMEM173 cDNA sequence artificially synthesized by eurofins Genomics was used. The amino acid sequence of mouse STING is shown in SEQ ID NO: 10 and the nucleotide sequence is shown in SEQ ID NO: 11. The synthesized cDNA was inserted into pET15b, an E. coli expression vector, using an In-Fusion HD Cloning kit to have a 6xHis tag composed of histidine 6 bases at the N terminus, an Avidin tag, and a TEV protease cleavage site, and pET15b-HisAviTEV-mSTING ( 138-341) expression plasmid of mouse wild type was constructed.

pCDF＿Duet-1 벡터에 인공 합성한 대장균 BirA (UniProt entry P06709) cDNA 를 삽입하고, pCDF＿Duet-1 BirA (1-321) 발현 플라스미드를 구축하였다.The artificially synthesized Escherichia coli BirA (UniProt entry P06709) cDNA was inserted into the pCDF_Duet-1 vector to construct a pCDF_Duet-1 BirA (1-321) expression plasmid.

(ii) STING C말 결합 도메인 단백질의 조제법 (ii) Preparation method of STING C-terminal binding domain protein

제조한 각 pET15b-HisAviTEV-hSTING (139-342) (인간 야생형, 인간 REF 변이형 및 인간 AQ 변이형의 STING C말 결합 도메인 단백질) 발현 플라스미드 및 pET15b-HisAviTEV-mSTING (138-341) (마우스 야생형의 STING C말 결합 도메인 단백질) 발현 플라스미드는 각각 pCDF＿Duet-1 BirA (1-321) 발현 플라스미드와 동시에 Competent E. coli Rosetta 2 (DE3) (Merck Millipore, MA, US) 로 형질 전환하고, 각 HisAviTEV-STING 발현주를 제조하였다. 이들 발현주를 100 ㎍/㎖ 암피실린, 50 ㎍/㎖ 스트렙토마이신, 및 30 ㎍/㎖ 카나마이신을 포함하는 TB 배지에 첨가하고, 37 ℃ 에서 배양 후, 100 μM IPTG 로 유도 발현하고 추가로 16 ℃ 에서 배양하였다.pET15b-HisAviTEV-hSTING (139-342) (STING C-terminal binding domain protein of human wild type, human REF variant and human AQ variant) expression plasmids and pET15b-HisAviTEV-mSTING (138-341) (mouse wild type) STING C-terminal binding domain protein) expression plasmids were transformed into Competent E. coli Rosetta 2 (DE3) (Merck Millipore, MA, US) simultaneously with pCDF_Duet-1 BirA (1-321) expression plasmids, respectively, and each HisAviTEV- A STING expression strain was prepared. These expression strains were added to TB medium containing 100 μg/mL ampicillin, 50 μg/mL streptomycin, and 30 μg/mL kanamycin, cultured at 37°C, induced expression with 100 μM IPTG, and further incubated at 16°C. cultured.

배양액을 원심하고, 얻어진 균체를 50 mM HEPES pH 8.0, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, 5 ％ (w/v) glycerol, Complete EDTA free 에 현탁 후, 동결 융해하였다. Lysozyme, DNase I 을 첨가한 후, 초음파 파쇄에 의해 단백질을 추출하고, 원심을 거쳐 상청을 회수하였다. 얻어진 상청을 AKTAexpress 크로마토그래피 시스템 (GE Healthcare, IL, US) 을 사용하여 HisTrap FF 칼럼 (GE Healthcare) 으로 정제하고, Superdex200 16/60 칼럼 (GE Healthcare) 에 통과시켜 Buffer (20 mM HEPES pH 7.5, 120 mM NaCl, 20 ％ Glycerol, 0.8 mM DTT) 로 용출하였다. SEC 로 목적 분자량의 단백질을 포함하는 획분 회수하고, His-Avi-TEV-hSTING (139-342) 인간 야생형 단백질, HisAviTEV-hSTING (139-342) 인간 REF 변이형 단백질, HisAviTEV-hSTING (139-342) 인간 AQ 변이형 단백질 및 His-Avi-TEV-mSTING (138-341) 마우스 야생형 단백질로서 회수하였다. 단백질 농도는 Nanodrop2000 (Thermo Fisher Scientific, MA, US) 을 사용하여 측정하고, 사용까지 -80 ℃ 에서 동결 보존하였다.The culture solution was centrifuged, and the obtained cells were suspended in 50 mM HEPES pH 8.0, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, 5% (w/v) glycerol, Complete EDTA free, and then frozen and thawed. After adding Lysozyme and DNase I, proteins were extracted by sonication, and the supernatant was collected by centrifugation. The obtained supernatant was purified with a HisTrap FF column (GE Healthcare) using an AKTAexpress chromatography system (GE Healthcare, IL, US), passed through a Superdex200 16/60 column (GE Healthcare), and Buffer (20 mM HEPES pH 7.5, 120 mM NaCl, 20% Glycerol, 0.8 mM DTT). Fractions containing the protein of the target molecular weight were recovered by SEC, and His-Avi-TEV-hSTING (139-342) human wild-type protein, HisAviTEV-hSTING (139-342) human REF mutant protein, HisAviTEV-hSTING (139-342 ) as human AQ variant protein and His-Avi-TEV-mSTING (138-341) mouse wild-type protein. Protein concentration was determined using Nanodrop2000 (Thermo Fisher Scientific, MA, US) and cryopreserved at -80°C until use.

HisAviTEV-hSTING (139-342) 인간 야생형 단백질의 아미노산 서열을 서열 번호 22, HisAviTEV-hSTING (139-342) 인간 REF 변이형 단백질의 아미노산 서열을 서열 번호 23, HisAviTEV-hSTING (139-342) 인간 AQ 변이형 단백질의 아미노산 서열을 서열 번호 24, His-Avi-TEV-mSTING (138-341) 마우스 야생형 단백질의 아미노산 서열을 서열 번호 25 에 나타낸다.HisAviTEV-hSTING (139-342) amino acid sequence of human wild-type protein SEQ ID NO: 22 HisAviTEV-hSTING (139-342) amino acid sequence of human REF variant protein SEQ ID NO: 23 HisAviTEV-hSTING (139-342) human AQ SEQ ID NO: 24 shows the amino acid sequence of the mutant protein, and SEQ ID NO: 25 shows the amino acid sequence of the His-Avi-TEV-mSTING (138-341) mouse wild-type protein.

(iii) STING 결합 시험 (iii) STING binding test

STING C말 결합 도메인 단백질에 대한 화합물의 결합성은, 단백질의 열 변성 온도의 상승을 지표로 하는 Protein thermal shift assay 에 의해 실시하였다.The binding of the compound to the STING C-terminal binding domain protein was performed by a protein thermal shift assay using an increase in the thermal denaturation temperature of the protein as an index.

상세하게는, 384 웰의 리얼타임 PCR 플레이트에, 어세이 버퍼 (20 mM Tris-HCl pH 7.5, 120 mM NaCl) 를 사용하여, 3 ㎕ 의 피험 화합물 (종농도 0.5 mM), 3 ㎕ 의 SYPRO Orange Protein Gel Stain (Thermo Fisher Scientific) (종농도 20 × 농도) 와 6 ㎕ 의 STING 단백질을 혼합하고, 플레이트 쉐이커를 사용하여 혼화하였다. 리얼타임 PCR 시스템 (Thermo Fisher Scientific) 에 의해, 온도를 매초 0.03 ℃ 의 속도로 25 ℃ 에서 95 ℃ 까지 상승시켜, 단백질의 열 변성 온도를 SYPRO Orange 가 발하는 형광을 지표로 측정하였다. 얻어진 측정값은, 해석 소프트웨어인 Protein Thermal Shift software (Thermo Fisher Scientific) 를 사용하여, 형광 강도의 증가 속도가 최대값을 나타내는 온도로서 Tm (the midpoint of the unfolding transition) (℃) 을 구하였다. 각 화합물의 Tm 값으로부터, 화합물 무첨가 웰의 Tm 값을 뺌으로써, 피험 화합물에 의한 Tm 의 시프트를 ΔTm (℃) 으로서 산출하였다. STING 단백질에 대한 결합 시험의 결과를 표 2 에 나타낸다.Specifically, in a 384-well real-time PCR plate, using assay buffer (20 mM Tris-HCl pH 7.5, 120 mM NaCl), 3 μl of the test compound (final concentration: 0.5 mM), 3 μl of SYPRO Orange Protein Gel Stain (Thermo Fisher Scientific) (final concentration 20 × concentration) and 6 μl of STING protein were mixed and mixed using a plate shaker. Using a real-time PCR system (Thermo Fisher Scientific), the temperature was raised from 25°C to 95°C at a rate of 0.03°C per second, and the heat denaturation temperature of the protein was measured using fluorescence emitted by SYPRO Orange as an indicator. For the obtained measured value, the midpoint of the unfolding transition (Tm) (°C) was determined as the temperature at which the fluorescence intensity increase rate exhibited the maximum value using protein thermal shift software (Thermo Fisher Scientific), which is an analysis software. By subtracting the Tm value of the compound-free well from the Tm value of each compound, the Tm shift by the test compound was calculated as ΔTm (°C). The results of the binding test for STING protein are shown in Table 2.

[표 2][Table 2]

Figure pat00634
Figure pat00634

이 결과, 본원 화합물이 인간의 야생형 STING 및 변이형 STING, 마우스의 야생형 STING 에 대하여 결합 활성을 갖는 것이 분명해졌다.As a result, it became clear that the present compound has binding activity to human wild-type STING and mutant STING, and mouse wild-type STING.

(시험예 3) 항종양 시험 (1) (Test Example 3) Antitumor test (1)

마우스 : 5 주령의 자성 (雌性) BALB/c 마우스 (BALB/cAnNCrlCrlj) (일본 찰스·리버) 를 실험 사용 전에 SPF 조건하에서 4 일간 이상 순화하였다.Mice: 5-week-old female BALB/c mice (BALB/cAnNCrlCrlj) (Charles River, Japan) were acclimatized under SPF conditions for 4 days or more before experimental use.

측정, 계산식 : 모든 연구에 있어서, 종양의 장경 및 단경을 전자식 디지털 캘리퍼 (CD15-CX, 미츠토요) 로 1 주간에 2 ∼ 3 회 측정하고, 종양 체적 (㎣) 을 계산하였다. 계산식은 이하에 나타내는 바와 같음. Measurement and calculation formula: In all studies, the major and minor diameters of tumors were measured two to three times a week with an electronic digital caliper (CD15-CX, Mitutoyo), and the tumor volume (mm 3 ) was calculated. Calculation formula is as showing below.

종양 체적 (㎣) = 0.5 × 장경 (㎜) × [단경 (㎜)]2 Tumor volume (mm) = 0.5 x major diameter (mm) x [short diameter (mm)] 2

시험 화합물은 생리 식염수 (오츠카 제약 공장) 에 의해 희석하여 사용하였다. 투여 시에는 50 ㎕ 를 종양내 투여하였다.The test compound was used after being diluted with physiological saline (Otsuka Pharmaceutical Co., Ltd.). At the time of administration, 50 μl was administered intratumorally.

American Type Culture Collection 사로부터 구입한 마우스 대장암 세포주 CT26.WT (CRL2638) 세포를 사용하였다. CT26.WT 세포를 생리 식염수에 현탁하고, 1.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 시험 화합물은 10 ㎍ 의 용량으로, Day 7, 9 및 11 에 합계 3 회 종양내 투여하였다. 또 비히클군으로서 생리 식염수를 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 5 마리 또는 6 마리로 하였다. Mouse colorectal cancer cell line CT26.WT (CRL2638) cells purchased from American Type Culture Collection were used. CT26.WT cells were suspended in physiological saline, and 1.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each test compound was intratumorally administered a total of 3 times on Days 7, 9 and 11 at a dose of 10 μg. Also, as a vehicle group, a group to which physiological saline was administered was set. The number of mice in each group was 5 or 6.

결과를 도 7a 및 도 7b 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 사각선은 화합물 번호 6a 투여군, 백색 역삼각선은 화합물 번호 8b 투여군, 백색 동그라미선은 화합물 번호 9b 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 화합물 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIGS. 7A and 7B. In the figure, the black square line indicates the vehicle group, the white square line indicates the Compound No. 6a administration group, the white inverted triangle indicates the Compound No. 8b administration group, and the open circle line indicates the Compound No. 9b administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. In contrast, in the compound-administered group, tumor growth was remarkably suppressed.

이상으로부터, 고리형 디뉴클레오티드 유도체의 종양내 투여에 의한 항종양 효과가 확인되었다.From the above, the antitumor effect of the intratumoral administration of the cyclic dinucleotide derivative was confirmed.

(시험예 4) 항종양 시험 (2) (Test Example 4) Antitumor test (2)

American Type Culture Collection 사로부터 구입한 마우스 대장암 세포주 CT26.WT (CRL2638) 에 인간 HER2 유전자를 도입한 CT26.WT-hHER2 세포를 만들었다. 상세하게는, 인간 HER2 의 cDNA 를 포함하는 플라스미드 (Clone ID IOH82145 ; Thermo Fisher Scientific) 를 사용하여 cDNA 를 증폭하고, 이것을 In-Fusion HD Cloning 키트 (Clontech) 를 사용하여 pQCXIN 레트로바이러스 벡터 (다카라 바이오) 에 삽입하였다. 인간 HER2 삽입 pQCXIN 레트로바이러스 벡터를 EcoPack2-293 세포주 (다카라 바이오) 에 Lipofectamine 3000 (Thermo Fisher Scientific) 을 사용하여 도입하고, 상청을 회수하여 CT26.WT 에 감염시켰다. 세포는 250 ㎍/㎖ Geneticin (Thermo Fisher Scientific) 첨가 배지에서 유지하였다.CT26.WT-hHER2 cells were prepared by introducing the human HER2 gene into the mouse colorectal cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection. Specifically, cDNA was amplified using a plasmid containing human HER2 cDNA (Clone ID IOH82145; Thermo Fisher Scientific), and this was amplified using an In-Fusion HD Cloning kit (Clontech) to pQCXIN retroviral vector (Takara Bio). inserted into. The human HER2 inserted pQCXIN retroviral vector was introduced into EcoPack2-293 cell line (Takara Bio) using Lipofectamine 3000 (Thermo Fisher Scientific), and the supernatant was recovered and infected CT26.WT. Cells were maintained in medium supplemented with 250 μg/ml Geneticin (Thermo Fisher Scientific).

항체 - CDN 콘쥬게이트는 아세트산염 완충액 (10 mM Acetate Buffer, 5 ％ Sorbitol, pH 5.5) (나칼라이 테스크) 에 의해 희석하여 사용하였다. 투여 시에는 200 ㎕ 를 꼬리 정맥내 투여하였다.The antibody-CDN conjugate was used after being diluted with an acetate buffer (10 mM Acetate Buffer, 5% Sorbitol, pH 5.5) (Nacalai Tesque). At the time of administration, 200 μl was administered intravenously in the tail vein.

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 30 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 8 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 7 at a dose of 30 μg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 8.

결과를 도 8 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은, 참고예 1 에서 제조한 개변 항HER2 항체에 실시예 8b 의 화합물을 콘쥬게이트시킨 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군, 흑색 삼각선은, 참고예 2 에서 제조한 개변 항LPS 항체에 동일하게 실시예 8b 의 화합물을 콘쥬게이트시킨 항LPS 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군 및 HER2 에 결합하지 않는 항LPS 항체 - CDN 콘쥬게이트 (1) 투여군에서는 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 8 . In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody-CDN conjugate (1) administration group in which the compound of Example 8b was conjugated to the modified anti-HER2 antibody prepared in Reference Example 1, and the black triangle indicates , Anti-LPS antibody-CDN conjugate (1) administration group in which the compound of Example 8b was conjugated to the modified anti-LPS antibody prepared in Reference Example 2 in the same way. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. Tumor growth progressed in the vehicle group and the anti-LPS antibody-CDN conjugate (1) administration group that does not bind to HER2. On the other hand, in the anti-HER2 antibody-CDN conjugate (1) administration group, tumor growth was remarkably suppressed.

이상으로부터, 항HER2 항체 - CDN 콘쥬게이트 (1) 의 정맥내 투여에 의한 항체 표적 의존적인 항종양 효과가 확인되었다.From the above, the antibody target-dependent antitumor effect by intravenous administration of the anti-HER2 antibody-CDN conjugate (1) was confirmed.

(시험예 5) 항종양 시험 (3) (Test Example 5) Antitumor test (3)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 6 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 30 ㎍ 의 용량으로, Day 6 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 6 마리 또는 8 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were subcutaneously transplanted into the right axilla of BALB/c mice (Day 0), and grouped at random after 6 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 6 at a dose of 30 µg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 6 or 8.

결과를 도 9 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 사각선은 항HER2 항체 - CDN 콘쥬게이트 (2) 투여군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (3) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 - CDN 콘쥬게이트 (2) 및 (3) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 9 . In the figure, the black square line indicates the vehicle group, the white square line indicates the anti-HER2 antibody-CDN conjugate (2) administration group, and the open triangle indicates the anti-HER2 antibody-CDN conjugate (3) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. On the other hand, in the anti-HER2 antibody-CDN conjugate (2) and (3) administration groups, tumor growth was remarkably suppressed.

이상으로부터, 항HER2 항체 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, the strong antitumor effect of the anti-HER2 antibody-CDN conjugate was confirmed.

(시험예 6) 항종양 시험 (4) (Test Example 6) Antitumor test (4)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 항체 - CDN 콘쥬게이트 (19) 는 30 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 6 마리로 하였다. CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. The antibody-CDN conjugate (19) was administered intravenously in the tail vein once on Day 7 at a dose of 30 µg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 6.

결과를 도 10 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (19) 투여군을 나타낸다. 항HER2 항체 - CDN 콘쥬게이트 (19) 는 약물 링커가 시스테인 콘쥬게이션으로 항체에 결합되어 있다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 - CDN 콘쥬게이트 (19) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 10 . The black square line in the figure represents the vehicle group, and the open triangle represents the anti-HER2 antibody-CDN conjugate (19) administration group. In the anti-HER2 antibody-CDN conjugate (19), a drug linker is linked to the antibody by cysteine conjugate. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. In contrast, in the anti-HER2 antibody-CDN conjugate (19)-administered group, tumor growth was remarkably suppressed.

이상으로부터, 항체와 약물 링커를 시스테인 콘쥬게이션 한 항HER2 항체 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, the strong antitumor effect of the anti-HER2 antibody-CDN conjugate obtained by conjugating the antibody and the drug linker with cysteine was confirmed.

(시험예 7) 항종양 시험 (5) (Test Example 7) Antitumor test (5)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 6 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 30 ㎍ 의 용량으로, Day 6 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 5 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were subcutaneously transplanted into the right axilla of BALB/c mice (Day 0), and grouped at random after 6 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 6 at a dose of 30 µg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 5.

결과를 도 11 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 - CDN 콘쥬게이트 (9) 투여군, 백색 역삼각선은 항HER2 항체 - CDN 콘쥬게이트 (10) 투여군, 백색 마름모꼴선은 항HER2 항체 - CDN 콘쥬게이트 (11) 투여군, 백색 동그라미선은 항HER2 항체 - CDN 콘쥬게이트 (12) 투여군, 백색 사각선은 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 항HER2 항체 - CDN 콘쥬게이트 (9), (10), (11), (12) 및 (1) 은 각각 상이한 링커로 실시예 8b 의 화합물이 콘쥬게이션 되어 있다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 - CDN 콘쥬게이트 (9), (10), (11) 및 (12) 투여군에서는 항HER2 항체 - CDN 콘쥬게이트 (1) 투여군과 마찬가지로 종양의 증식이 현저하게 억제되었다.The result is shown in FIG. 11. In the figure, the black square line is the vehicle group, the white triangle is the anti-HER2 antibody-CDN conjugate (9) administration group, the white inverted triangle is the anti-HER2 antibody-CDN conjugate (10) administration group, and the white diamond line is the anti-HER2 antibody-CDN The conjugate (11) administration group, the open circle line represents the anti-HER2 antibody-CDN conjugate (12) administration group, and the open square line represents the anti-HER2 antibody-CDN conjugate (1) administration group. Anti-HER2 antibody-CDN conjugates (9), (10), (11), (12) and (1) each have the compound of Example 8b conjugated with a different linker. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. In contrast, in the anti-HER2 antibody-CDN conjugate (9), (10), (11) and (12)-administered groups, tumor growth was markedly inhibited as in the anti-HER2 antibody-CDN conjugate (1)-administered group.

이상으로부터, 항HER2 항체 - CDN 콘쥬게이트는 링커를 바꾼 경우에 있어서도 항종양 효과를 나타내는 것이 확인되었다.From the above, it was confirmed that the anti-HER2 antibody-CDN conjugate exhibits an antitumor effect even when the linker is changed.

(시험예 8) 항종양 시험 (6) (Test Example 8) Antitumor test (6)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 투여 검체는, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 5 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each administration specimen was intravenously administered in the tail vein once on Day 7 in total. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 5.

결과를 도 12 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (1) 의 60 ㎍ 투여군, 흑색 역삼각선은 항HER2 항체 2 의 59 ㎍ 투여군, 흑색 동그라미선은 화합물 번호 8b 의 1.2 ㎍ 투여군을 나타낸다. 항HER2 항체 2 및 화합물 번호 8b 의 투여량은, 항HER2 항체 2 - CDN 콘쥬게이트 (1) 을 구성하는 각 성분의 당량이다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 2 - CDN 콘쥬게이트 (1) 투여군에서는 종양의 증식이 현저하게 억제되었다. 그러나, 항HER2 항체 2 및 화합물 번호 8b 의 당량을 투여했을 때는 종양의 증식을 억제하지 않았다.The results are shown in FIG. 12 . In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody 2-CDN conjugate (1) 60 μg administration group, the black inverted triangle indicates the anti-HER2 antibody 2 59 μg administration group, and the black dotted line indicates Compound No. 8b. The 1.2 μg administration group is shown. The doses of anti-HER2 antibody 2 and Compound No. 8b are equivalents of each component constituting the anti-HER2 antibody 2-CDN conjugate (1). The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. On the other hand, in the group administered with the anti-HER2 antibody 2-CDN conjugate (1), tumor growth was remarkably suppressed. However, tumor growth was not inhibited when equivalent amounts of anti-HER2 antibody 2 and Compound No. 8b were administered.

이상으로부터, 항HER2 항체 2 - CDN 콘쥬게이트 (1) 은 항종양 효과를 나타내고, 당량의 항HER2 항체 2 및 화합물 번호 8b 는, 꼬리 정맥내 투여에서는 항종양 효과를 나타내지 않는 것이 확인되었다.From the above, it was confirmed that the anti-HER2 antibody 2-CDN conjugate (1) exhibited an anti-tumor effect, and the anti-HER2 antibody 2 and the equivalent of Compound No. 8b did not exhibit an anti-tumor effect when administered intravenously in the tail vein.

(시험예 9) 항종양 시험 (7) (Test Example 9) Antitumor test (7)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 30 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 5 마리 또는 6 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 7 at a dose of 30 μg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 5 or 6.

결과를 도 13(a) 내지 (c) 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 각 백색 심볼선은 평가한 각 항HER2 항체 2 - CDN 콘쥬게이트 (2), (3), (4), (5), (6), (7) 및 (8) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 2 - CDN 콘쥬게이트 (2), (3), (4), (5), (6), (7) 및 (8) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in Figs. 13(a) to (c). In the figure, the black square line indicates the vehicle group, and each white symbol line indicates each of the evaluated anti-HER2 antibody 2-CDN conjugates (2), (3), (4), (5), (6), (7) and ( 8) Indicates the administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. On the other hand, in the anti-HER2 antibody 2-CDN conjugate (2), (3), (4), (5), (6), (7) and (8) administration groups, tumor growth was remarkably suppressed.

이상으로부터, 항HER2 항체 2 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, the strong antitumor effect of the anti-HER2 antibody 2-CDN conjugate was confirmed.

(시험예 10) 항종양 시험 (8) (Test Example 10) Antitumor test (8)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 60 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 5 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 7 at a dose of 60 μg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 5.

결과를 도 14 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (9) 투여군, 백색 동그라미선은 항HER2 항체 2 - CDN 콘쥬게이트 (10) 투여군을 나타낸다. 항HER2 항체 2 - CDN 콘쥬게이트 (9) 및 (10) 은 약물 평균 결합수가 약 2 인 MSG 형 당사슬 리모델링 항체를 사용한 항체 - CDN 콘쥬게이트이다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 2 - CDN 콘쥬게이트 (9) 및 (10) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 14 . In the figure, the black square line indicates the vehicle group, the open triangle indicates the anti-HER2 antibody 2-CDN conjugate (9) administration group, and the open circle line indicates the anti-HER2 antibody 2-CDN conjugate (10) administration group. Anti-HER2 antibody 2-CDN conjugates (9) and (10) are antibody-CDN conjugates using MSG-type sugar chain remodeling antibodies having an average drug binding number of about 2. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. On the other hand, in the anti-HER2 antibody 2-CDN conjugate (9) and (10) administration groups, tumor growth was remarkably suppressed.

이상으로부터, 약물 평균 결합수가 약 2 인 항HER2 항체 2 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, the strong antitumor effect of the anti-HER2 antibody 2-CDN conjugate having an average drug binding number of about 2 was confirmed.

(시험예 11) 항종양 시험 (9) (Test Example 11) Antitumor test (9)

American Type Culture Collection 사로부터 구입한 마우스 대장암 세포주 CT26.WT (CRL2638) 에 인간 EphA2 유전자를 도입한 CT26.WT-hEphA2 세포를 만들었다. 상세하게는, 인간 EphA2 의 cDNA 를 포함하는 pDONR221 (Thermo Fisher Scientific) 를 사용하여 cDNA 를 증폭하고, 이것을 Gateway vector conversion system (Thermo Fisher Scientific) 을 사용하여 pLNCX 레트로바이러스 벡터 (다카라 바이오) 에 삽입하였다. 인간 EphA2 삽입 pLNCX 레트로바이러스 벡터를 EcoPack2-293 세포주 (다카라 바이오) 에 Lipofectamine 2000 (Thermo Fisher Scientific) 을 사용하여 도입하고, 상청을 회수하여 CT26.WT 에 감염시켰다. 세포는 500 ㎍/㎖ Geneticin (Thermo Fisher Scientific) 첨가 배지에서 유지하였다.CT26.WT-hEphA2 cells were prepared by introducing the human EphA2 gene into the mouse colorectal cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection. Specifically, the cDNA was amplified using pDONR221 (Thermo Fisher Scientific) containing the cDNA of human EphA2, and inserted into a pLNCX retroviral vector (Takara Bio) using the Gateway vector conversion system (Thermo Fisher Scientific). The human EphA2 inserted pLNCX retroviral vector was introduced into the EcoPack2-293 cell line (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific), and the supernatant was recovered and infected into CT26.WT. Cells were maintained in medium supplemented with 500 μg/ml Geneticin (Thermo Fisher Scientific).

CT26.WT-hEphA2 세포를 인산 완충액에 현탁하고, 1.9 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 항EphA2 항체 및 항EphA2 항체 - CDN 콘쥬게이트 (1) 은 60 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 8 마리로 하였다.CT26.WT-hEphA2 cells were suspended in phosphate buffer, and 1.9 × 10 6 cells were subcutaneously transplanted into the right axillary region of BALB/c mice (Day 0), and grouped at random after 7 days. The anti-EphA2 antibody and the anti-EphA2 antibody-CDN conjugate (1) were administered intravenously in the tail vein once on Day 7 at a dose of 60 μg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 8.

결과를 도 15 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 동그라미선은 항EphA2 항체 투여군, 백색 삼각선은 항EphA2 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 항EphA2 항체 투여군은 종양의 증식을 억제하지 않았다. 이에 반해, 항EphA2 항체 - CDN 콘쥬게이트 (1) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 15 . The black square line in the figure represents the vehicle group, the open circle line represents the anti-EphA2 antibody administration group, and the open triangle represents the anti-EphA2 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. The anti-EphA2 antibody-administered group did not inhibit tumor growth. On the other hand, in the group administered with the anti-EphA2 antibody-CDN conjugate (1), tumor growth was remarkably suppressed.

이상으로부터, 항EphA2 항체가 항종양 효과를 나타내지 않는 모델을 사용하여, 항EphA2 항체 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, a strong anti-tumor effect of the anti-EphA2 antibody-CDN conjugate was confirmed using a model in which the anti-EphA2 antibody does not exhibit an anti-tumor effect.

(시험예 12) 항종양 시험 (10) (Test Example 12) Antitumor test (10)

American Type Culture Collection 사로부터 구입한 마우스 림프구형 세포주 P388D1 (CCL-46) 에 인간 CD33 유전자를 도입한 P388D1-hCD33 세포를 만들었다. 상세하게는, 인간 CD33 의 cDNA 를 삽입한 pLVSIN 렌티바이러스 벡터 (다카라 바이오) 를 만들고, Lenti-X293T 세포주 (다카라 바이오) 에 Lentiviral High Titer Packaging Mix (다카라 바이오) 를 사용하여 도입하고, 상청을 회수하여 P388D1 에 감염시켰다. 세포는 2 ㎍/㎖ Puromycin (Thermo Fisher Scientific) 첨가 배지에서 유지하였다.P388D1-hCD33 cells were prepared by introducing the human CD33 gene into the mouse lymphoid cell line P388D1 (CCL-46) purchased from American Type Culture Collection. Specifically, a pLVSIN lentiviral vector (Takara Bio) into which cDNA of human CD33 was inserted was created, introduced into the Lenti-X293T cell line (Takara Bio) using Lentiviral High Titer Packaging Mix (Takara Bio), and the supernatant was recovered, Infected with P388D1. Cells were maintained in medium supplemented with 2 μg/ml Puromycin (Thermo Fisher Scientific).

마우스는 4 주령의 자성 DBA/2 마우스 (DBA/2NCrl) (일본 찰스·리버) 를 구입하고, 실험 사용 전에 SPF 조건하에서 5 일간 순화하였다.As mice, 4-week-old female DBA/2 mice (DBA/2NCrl) (Charles River, Japan) were purchased and acclimatized for 5 days under SPF conditions before experimental use.

P388D1-hCD33 세포를 인산 완충액에 현탁하고, 1.0 × 106 세포를 DBA/2 마우스의 우측 액와부에 피하 이식하고 (Day 0), 4 일 후에 무작위로 군 분류를 실시하였다. 항CD33 항체 및 항CD33 항체 - CDN 콘쥬게이트 (1) 은 60 ㎍ 의 용량으로, Day 4 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 10 마리로 하였다.P388D1-hCD33 cells were suspended in phosphate buffer, and 1.0 × 10 6 cells were transplanted subcutaneously to the right axilla of DBA/2 mice (Day 0), and grouped randomly 4 days later. The anti-CD33 antibody and the anti-CD33 antibody-CDN conjugate (1) were administered intravenously in the tail vein once on Day 4 at a dose of 60 µg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 10.

결과를 도 16 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 동그라미선은 항CD33 항체 투여군, 백색 삼각선은 항CD33 항체 - CDN 콘쥬게이트 (1) 투여군을 나타낸다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 항CD33 항체 투여군은 종양의 증식을 억제하지 않았다. 이에 반해, 항CD33 항체 - CDN 콘쥬게이트 (1) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 16 . In the figure, the black square line indicates the vehicle group, the open circle line indicates the anti-CD33 antibody administration group, and the open triangle line indicates the anti-CD33 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. The anti-CD33 antibody administration group did not inhibit tumor growth. On the other hand, in the anti-CD33 antibody-CDN conjugate (1) administration group, tumor growth was remarkably suppressed.

이상으로부터, 항CD33 항체가 항종양 효과를 나타내지 않는 모델을 사용하여, 항CD33 항체 - CDN 콘쥬게이트의 강한 항종양 효과가 확인되었다.From the above, the strong antitumor effect of the anti-CD33 antibody-CDN conjugate was confirmed using a model in which the anti-CD33 antibody does not exhibit an anti-tumor effect.

(시험예 13) 항종양 시험 (11) (Test Example 13) Antitumor test (11)

CT26.WT-hHER2 세포를 생리 식염수에 현탁하고, 5.0 × 106 세포를 BALB/c 마우스의 우측 액와부에 피하 이식하고 (Day 0), 7 일 후에 무작위로 군 분류를 실시하였다. 각 항체 - CDN 콘쥬게이트는 60 ㎍ 의 용량으로, Day 7 에 합계 1 회 꼬리 정맥내 투여하였다. 또 비히클군으로서 아세트산염 완충액을 투여하는 군을 설정하였다. 각 군의 마우스 마릿수는 6 마리로 하였다.CT26.WT-hHER2 cells were suspended in physiological saline, and 5.0 × 10 6 cells were transplanted subcutaneously to the right axilla of BALB/c mice (Day 0), and grouping was performed at random after 7 days. Each antibody-CDN conjugate was administered intravenously in the tail vein once on Day 7 at a dose of 60 μg. Also, as a vehicle group, a group to which acetate buffer was administered was set. The number of mice in each group was 6.

결과를 도 22 에 나타낸다. 도면 중의 흑색 사각선은 비히클군, 백색 삼각선은 항HER2 항체 2 - CDN 콘쥬게이트 (11) 투여군, 백색 동그라미선은 항HER2 항체 2 - CDN 콘쥬게이트 (12) 투여군을 나타낸다. 항HER2 항체 2 - CDN 콘쥬게이트 (11) 및 (12) 에 콘쥬게이션 되어 있는 화합물 번호 52a 및 52b 는 인산기를 갖는 CDN 이다. 세로축은 종양 체적 (㎣), 가로축은 종양 이식 후의 일수를 나타내고 있다. 비히클군은 종양의 증식이 진행되었다. 이에 반해, 항HER2 항체 2 - CDN 콘쥬게이트 (11) 및 (12) 투여군에서는 종양의 증식이 현저하게 억제되었다.The results are shown in FIG. 22 . In the figure, the black square line indicates the vehicle group, the white triangle indicates the anti-HER2 antibody 2-CDN conjugate (11) administration group, and the open circle line indicates the anti-HER2 antibody 2-CDN conjugate (12) administration group. Compound Nos. 52a and 52b conjugated to the anti-HER2 antibody 2-CDN conjugates (11) and (12) are CDNs having a phosphate group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. In the vehicle group, tumor growth progressed. In contrast, in the anti-HER2 antibody 2-CDN conjugate (11) and (12) administration groups, tumor growth was remarkably suppressed.

이상으로부터, 인산기를 갖는 CDN 을 콘쥬게이트 한 항HER2 항체 2 - CDN 콘쥬게이트 (11) 및 (12) 의 강한 항종양 효과가 확인되었다.From the above, strong antitumor effects of anti-HER2 antibody 2-CDN conjugates (11) and (12) conjugated with CDN having a phosphate group were confirmed.

본 발명에 의해 강한 STING 아고니스트 활성을 갖고, 강한 항종양 효과를 나타내는 신규 CDN 유도체가 제공된다. 또, 본 발명에 의해 당해 신규 CDN 유도체를 포함하는 항체 약물 콘쥬게이트가 제공된다. 이들은, STING 아고니스트 활성이 관련하는 질환 (예를 들어, 암) 의 치료제로서 유용하다.According to the present invention, a novel CDN derivative having a strong STING agonist activity and exhibiting a strong antitumor effect is provided. In addition, antibody drug conjugates containing the novel CDN derivatives are provided by the present invention. These are useful as therapeutic agents for diseases (for example, cancer) in which STING agonist activity is involved.

서열표 프리텍스트sequence table free text

서열 번호 1 : 트라스투주맙의 경사슬의 아미노산 서열 SEQ ID NO: 1: Amino acid sequence of the light chain of Trastuzumab

서열 번호 2 : 트라스투주맙의 중사슬의 아미노산 서열 SEQ ID NO: 2: Amino acid sequence of the heavy chain of Trastuzumab

서열 번호 3 : 개변 항HER2 항체의 중사슬의 아미노산 서열 SEQ ID NO: 3: amino acid sequence of the heavy chain of the modified anti-HER2 antibody

서열 번호 4 : 인간 H232 (REF) 변이형 STING 의 아미노산 서열 SEQ ID NO: 4: amino acid sequence of human H232 (REF) variant STING

서열 번호 5 : 인간 H232 (REF) 변이형 STING 의 뉴클레오티드 서열 SEQ ID NO: 5: Nucleotide sequence of human H232 (REF) variant STING

서열 번호 6 : 인간 야생형 STING 의 아미노산 서열 SEQ ID NO: 6: amino acid sequence of human wild-type STING

서열 번호 7 : 인간 야생형 STING 의 뉴클레오티드 서열 SEQ ID NO: 7: Nucleotide sequence of human wild-type STING

서열 번호 8 : 인간 HAQ 변이형 STING 의 아미노산 서열 SEQ ID NO: 8: amino acid sequence of human HAQ variant STING

서열 번호 9 : 인간 HAQ 변이형 STING 의 뉴클레오티드 서열 SEQ ID NO: 9: Nucleotide sequence of human HAQ variant STING

서열 번호 10 : 마우스 STING 의 아미노산 서열 SEQ ID NO: 10: amino acid sequence of mouse STING

서열 번호 11 : 마우스 STING 의 뉴클레오티드 서열 SEQ ID NO: 11: Nucleotide sequence of mouse STING

서열 번호 12 ∼ 21 : 프라이머의 서열 SEQ ID NOs: 12 to 21: primer sequence

서열 번호 22 : HisAviTEV-hSTING (139-342) 인간 야생형 단백질의 아미노산 서열 SEQ ID NO: 22: amino acid sequence of HisAviTEV-hSTING (139-342) human wild-type protein

서열 번호 23 : HisAviTEV-hSTING (139-342) 인간 REF 변이형 단백질의 아미노산 서열 SEQ ID NO: 23: amino acid sequence of HisAviTEV-hSTING (139-342) human REF variant protein

서열 번호 24 : HisAviTEV-hSTING (139-342) 인간 AQ 변이형 단백질의 아미노산 서열 SEQ ID NO: 24: amino acid sequence of HisAviTEV-hSTING (139-342) human AQ variant protein

서열 번호 25 : His-Avi-TEV-mSTING (138-341) 마우스 야생형 단백질의 아미노산 서열 SEQ ID NO: 25: amino acid sequence of His-Avi-TEV-mSTING (138-341) mouse wild-type protein

서열 번호 26 : 개변 항LPS 항체의 경사슬의 아미노산 서열 SEQ ID NO: 26: amino acid sequence of the light chain of the modified anti-LPS antibody

서열 번호 27 : 개변 항LPS 항체의 중사슬의 아미노산 서열 SEQ ID NO: 27: amino acid sequence of the heavy chain of the modified anti-LPS antibody

서열 번호 28 : 페르투주맙 경사슬의 아미노산 서열 SEQ ID NO: 28: amino acid sequence of Pertuzumab light chain

서열 번호 29 : 페르투주맙 중사슬의 아미노산 서열 SEQ ID NO: 29: amino acid sequence of Pertuzumab heavy chain

서열 번호 30 : 개변 항HER2 항체 2 의 중사슬의 아미노산 서열 SEQ ID NO: 30: Amino acid sequence of heavy chain of modified anti-HER2 antibody 2

서열 번호 31 : 항CD33 항체 경사슬의 아미노산 서열 SEQ ID NO: 31: amino acid sequence of anti-CD33 antibody light chain

서열 번호 32 : 항CD33 항체 중사슬의 아미노산 서열 SEQ ID NO: 32: amino acid sequence of anti-CD33 antibody heavy chain

서열 번호 33 : 항EphA2 항체 경사슬의 아미노산 서열 SEQ ID NO: 33: amino acid sequence of anti-EphA2 antibody light chain

서열 번호 34 : 항EphA2 항체 중사슬의 아미노산 서열 SEQ ID NO: 34: amino acid sequence of anti-EphA2 antibody heavy chain

서열 번호 35 : 항CDH6 항체 경사슬의 아미노산 서열 SEQ ID NO: 35: amino acid sequence of anti-CDH6 antibody light chain

서열 번호 36 : 항CDH6 항체 중사슬의 아미노산 서열SEQ ID NO: 36: amino acid sequence of anti-CDH6 antibody heavy chain

SEQUENCE LISTING <110> DAIICHI SANKYO COMPANY, LIMITED <120> Novel Cyclic Dinucleotide Derivatives and Antibody-Drug Conjugate thereof <130> SAP-855-PCT <141> 2019-09-06 <150> JP 2018167369 <151> 2018-09-06 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab Light Chain <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab Heavy Chain <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 3 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Modified HER2 antibody Heavy Chain <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 4 <211> 379 <212> PRT <213> Homo sapiens <400> 4 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys 210 215 220 Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr 225 230 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 5 <211> 1140 <212> DNA <213> Homo sapiens <400> 5 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60 gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 ttcctggata aactgcccca gcagaccggt gaccatgctg gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 6 <211> 379 <212> PRT <213> Homo sapiens <400> 6 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys 210 215 220 Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr 225 230 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 7 <211> 1140 <212> DNA <213> Homo sapiens <400> 7 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60 gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 ttcctggata aactgcccca gcagaccggt gaccgtgctg gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 8 <211> 379 <212> PRT <213> Homo sapiens <400> 8 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu His His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys 210 215 220 Leu Pro Gln Gln Thr Ala Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr 225 230 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Gln Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 9 <211> 1140 <212> DNA <213> Homo sapiens <400> 9 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60 gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg caccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 ttcctggata aactgcccca gcagaccgct gaccgtgctg gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccaga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 10 <211> 378 <212> PRT <213> Mus musculus <400> 10 Met Pro Tyr Ser Asn Leu His Pro Ala Ile Pro Arg Pro Arg Gly His 1 5 10 15 Arg Ser Lys Tyr Val Ala Leu Ile Phe Leu Val Ala Ser Leu Met Ile 20 25 30 Leu Trp Val Ala Lys Asp Pro Pro Asn His Thr Leu Lys Tyr Leu Ala 35 40 45 Leu His Leu Ala Ser His Glu Leu Gly Leu Leu Leu Lys Asn Leu Cys 50 55 60 Cys Leu Ala Glu Glu Leu Cys His Val Gln Ser Arg Tyr Gln Gly Ser 65 70 75 80 Tyr Trp Lys Ala Val Arg Ala Cys Leu Gly Cys Pro Ile His Cys Met 85 90 95 Ala Met Ile Leu Leu Ser Ser Tyr Phe Tyr Phe Leu Gln Asn Thr Ala 100 105 110 Asp Ile Tyr Leu Ser Trp Met Phe Gly Leu Leu Val Leu Tyr Lys Ser 115 120 125 Leu Ser Met Leu Leu Gly Leu Gln Ser Leu Thr Pro Ala Glu Val Ser 130 135 140 Ala Val Cys Glu Glu Lys Lys Leu Asn Val Ala His Gly Leu Ala Trp 145 150 155 160 Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Gly Leu Gln Ala 165 170 175 Arg Ile Arg Met Phe Asn Gln Leu His Asn Asn Met Leu Ser Gly Ala 180 185 190 Gly Ser Arg Arg Leu Tyr Ile Leu Phe Pro Leu Asp Cys Gly Val Pro 195 200 205 Asp Asn Leu Ser Val Val Asp Pro Asn Ile Arg Phe Arg Asp Met Leu 210 215 220 Pro Gln Gln Asn Ile Asp Arg Ala Gly Ile Lys Asn Arg Val Tyr Ser 225 230 235 240 Asn Ser Val Tyr Glu Ile Leu Glu Asn Gly Gln Pro Ala Gly Val Cys 245 250 255 Ile Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln 260 265 270 Asp Ala Lys Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys 275 280 285 Leu Phe Cys Arg Thr Leu Glu Glu Ile Leu Glu Asp Val Pro Glu Ser 290 295 300 Arg Asn Asn Cys Arg Leu Ile Val Tyr Gln Glu Pro Thr Asp Gly Asn 305 310 315 320 Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Ile Arg Gln Glu Glu 325 330 335 Lys Glu Glu Val Thr Met Asn Ala Pro Met Thr Ser Val Ala Pro Pro 340 345 350 Pro Ser Val Leu Ser Gln Glu Pro Arg Leu Leu Ile Ser Gly Met Asp 355 360 365 Gln Pro Leu Pro Leu Arg Thr Asp Leu Ile 370 375 <210> 11 <211> 1137 <212> DNA <213> Mus musculus <400> 11 atgccatact ccaacctgca tccagccatc ccacggccca gaggtcaccg ctccaaatat 60 gtagccctca tctttctggt ggccagcctg atgatccttt gggtggcaaa ggatccacca 120 aatcacactc tgaagtacct agcacttcac ctagcctcgc acgaacttgg actactgttg 180 aaaaacctct gctgtctggc tgaagagctg tgccatgtcc agtccaggta ccagggcagc 240 tactggaagg ctgtgcgcgc ctgcctggga tgccccatcc actgtatggc tatgattcta 300 ctatcgtctt atttctattt cctccaaaac actgctgaca tatacctcag ttggatgttt 360 ggccttctgg tcctctataa gtccctaagc atgctcctgg gccttcagag cttgactcca 420 gcggaagtct ctgcagtctg tgaagaaaag aagttaaatg ttgcccacgg gctggcctgg 480 tcatactaca ttgggtactt gcggttgatc ttaccagggc tccaggcccg gatccgaatg 540 ttcaatcagc tacataacaa catgctcagt ggtgcaggga gccgaagact gtacatcctc 600 tttccattgg actgtggggt gcctgacaac ctgagtgtag ttgaccccaa cattcgattc 660 cgagatatgc tgccccagca aaacatcgac cgtgctggca tcaagaatcg ggtttattcc 720 aacagcgtct acgagattct ggagaacgga cagccagcag gcgtctgtat cctggagtac 780 gccaccccct tgcagaccct gtttgccatg tcacaggatg ccaaagctgg cttcagtcgg 840 gaggatcggc ttgagcaggc taaactcttc tgccggacac ttgaggaaat cctggaagat 900 gtccccgagt ctcgaaataa ctgccgcctc attgtctacc aagaacccac agacggaaac 960 agtttctcac tgtctcagga ggtgctccgg cacattcgtc aggaagaaaa ggaggaggtt 1020 accatgaatg cccccatgac ctcagtggca cctcctccct ccgtactgtc ccaagagcca 1080 agactcctca tcagtggtat ggatcagcct ctcccactcc gcactgacct catctga 1137 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cgtgctggca tcaaggatcg ggtttac 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gtcaccggtc tgctggggca gtttatc 27 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctgaccgtg ctggcatcaa ggatcgggtt tac 33 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ggtctgctgg ggcagtttat ccagg 25 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 caccacatcc actccaggta ccgg 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cagctcctca gccaggctgc agac 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cagacacttg aggacatcct ggcag 25 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcagaagagt ttggcctgct caa 23 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 acctgtattt tcagggcctg gccccagctg agatctctg 39 <210> 21 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cagaattcgc aagcttttaa gtaacctctt ccttttcctc ctgc 44 <210> 22 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) human WT <400> 22 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 23 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) human REF mutant <400> 23 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 24 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) human AQ mutant <400> 24 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Ala Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Gln Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 25 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) mouse WT <400> 25 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Thr Pro Ala Glu Val Ser Ala Val 35 40 45 Cys Glu Glu Lys Lys Leu Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Gly Leu Gln Ala Arg Ile 65 70 75 80 Arg Met Phe Asn Gln Leu His Asn Asn Met Leu Ser Gly Ala Gly Ser 85 90 95 Arg Arg Leu Tyr Ile Leu Phe Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Val Val Asp Pro Asn Ile Arg Phe Arg Asp Met Leu Pro Gln 115 120 125 Gln Asn Ile Asp Arg Ala Gly Ile Lys Asn Arg Val Tyr Ser Asn Ser 130 135 140 Val Tyr Glu Ile Leu Glu Asn Gly Gln Pro Ala Gly Val Cys Ile Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Asp Ala 165 170 175 Lys Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Glu Ile Leu Glu Asp Val Pro Glu Ser Arg Asn 195 200 205 Asn Cys Arg Leu Ile Val Tyr Gln Glu Pro Thr Asp Gly Asn Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Ile Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 26 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Modified LPS Antibody Light Chain <400> 26 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ala Ser Glu Asn Val Gly Asn Ser 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr His Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 27 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Modified LPS Antibody Heavy Chain <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Gly Thr Arg Ser Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Val Tyr Tyr Asp His Val Gly Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab Light Chain <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab Heavy Chain <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 30 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Modified HER2 Antibody2 Heavy Chain <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 31 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD33 Antibody Light Chain <400> 31 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr 20 25 30 Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 32 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD33 Antibody Heavy Chain <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 33 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Anti-EphA2 Antibody Light Chain <400> 33 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 34 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Anti-EphA2 Antibody Heavy Chain <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 35 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Anti-CDH6 Antibody Light Chain <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Tyr Lys Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Ala 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 36 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Anti-CDH6 Antibody Heavy Chain <400> 36 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Phe Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Glu Thr Glu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Tyr Gly Gly Phe Ala Gly Gly Tyr Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 SEQUENCE LISTING <110> DAIICHI SANKYO COMPANY, LIMITED <120> Novel Cyclic Dinucleotide Derivatives and Antibody-Drug Conjugate Its <130> SAP-855-PCT <141> 2019-09-06 <150> JP 2018167369 <151> 2018-09 -06 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab Light Chain <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab Heavy Chain <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val S er Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 3 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Modified HER2 antibody Heavy Chain <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Ty r Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 4 <211> 379 <212> PRT <213> Homo sapiens <400> 4 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu A sp Lys 210 215 220 Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr 225 230 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu L eu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 5 <211> 1140 <212> DNA <213> Homo sapiens <400> 5 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg 6t gccagaaggg gg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 gcacctgcccat gactgcctgccata aactgcctgccata gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 6 <211> 379 <212> PRT <213> Homo sapiens <400> 6 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys 210 215 220 Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr 225 23 0 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 7 <211> 1140 <212> DNA <213> Homo sapiens <400> 7 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60 gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 ttcctggata aactgcccca gcagaccggt gaccgtgctg gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcaca at acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 8 <211> 379 <212> PRT <213> Homo sapiens <400> 8 Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His 1 5 10 15 Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr 20 25 30 Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val 35 40 45 Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Leu Asn Gly Val Cys 50 55 60 Ser Leu Ala Glu Glu Leu His His Ile His Ser Arg Tyr Arg Gly Ser 65 70 75 80 Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly 85 90 95 Ala Leu Leu Leu Leu Ser Ile Tyr Tyr Tyr Ser Leu Pro Asn Ala 100 105 110 Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln 115 120 125 Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile 130 135 140 Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala 145 150 155 160 Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln 165 170 175 Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly 180 185 190 Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val 195 200 205 Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys 210 215 220 Leu Pro Gln Gln Thr Ala Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr 225 230 235 240 Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr 245 250 255 Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser 260 265 270 Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala 275 280 285 Lys Leu Phe Cys Gln Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu 290 295 300 Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp 305 310 315 320 Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu 325 330 335 Glu Lys Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro 340 345 350 Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met 355 360 365 Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser 370 375 <210> 9 <211> 1140 <212> DNA <213> Homo sapiens <400> 9 atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60 gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120 gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180 aacggggtct gcagcctggc tgaggagctg caccacatcc actccaggta ccggggcagc 240 tactggagga ctgtgcgggc ctg cctgggc tgccccctcc gccgtggggc cctgttgctg 300 ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360 cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420 ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480 tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540 acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600 ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660 ttcctggata aactgcccca gcagaccgct gaccgtgctg gcatcaagga tcgggtttac 720 agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780 tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840 cgggaggata ggcttgagca ggccaaactc ttctgccaga cacttgagga catcctggca 900 gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960 agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020 gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080 cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140 <210> 10 <211> 378 <212> PRT <213> Mus musculus <400> 10 Met Pro Tyr Ser Asn Leu His Pro Ala Ile Pro Arg Pro Arg Gly His 1 5 10 15 Arg Ser Lys Tyr Val Ala Leu Ile Phe Leu Val Ala Ser Leu Met Ile 20 25 30 Leu Trp Val Ala Lys Asp Pro Pro Asn His Thr Leu Lys Tyr Leu Ala 35 40 45 Leu His Leu Ala Ser His Glu Leu Gly Leu Leu Leu Lys Asn Leu Cys 50 55 60 Cys Leu Ala Glu Glu Leu Cys His Val Gln Ser Arg Tyr Gln Gly Ser 65 70 75 80 Tyr Trp Lys Ala Val Arg Ala Cys Leu Gly Cys Pro Ile His Cys Met 85 90 95 Ala Met Ile Leu Leu Ser Ser Tyr Phe Tyr Phe Leu Gln Asn Thr Ala 100 105 110 Asp Ile Tyr Leu Ser Trp Met Phe Gly Leu Leu Val Leu Tyr Lys Ser 115 120 125 Leu Ser Met Leu Leu Gly Leu Gln Ser Leu Thr Pro Ala Glu Val Ser 130 135 140 Ala Val Cys Glu Glu Lys Lys Leu Asn Val Ala His Gly Leu Ala Trp 145 150 155 160 Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Gly Leu Gln Ala 165 170 175 Arg Ile Arg Met Phe Asn Gln Leu His Asn Asn Met Leu Ser Gly Ala 180 185 190 Gly Ser Arg Arg Leu Tyr Ile Leu Phe Pro Leu Asp Cys Gly Val Pro 195 200 205 Asp Asn Leu Ser Val Val Asp Pro Asn Ile Arg Phe Arg Asp Met Leu 210 215 220 Pro Gln Gln Asn Ile Asp Arg Ala Gly Ile Lys Asn Arg Val Tyr Ser 225 230 235 240 Asn Ser Val Tyr Glu Ile Leu Glu Asn Gly Gln Pro Ala Gly Val Cys 245 250 255 Ile Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln 260 265 270 Asp Ala Lys Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys 275 280 285 Leu Phe Cys Arg Thr Leu Glu Glu Ile Leu Glu Asp Val Pro Glu Ser 290 295 300 Arg Asn Asn Cys Arg Leu Ile Val Tyr Gln Glu Pro Thr Asp Gly Asn 305 310 315 320 Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Ile Arg Gln Glu Glu 325 330 335 Lys Glu Glu Val Thr Met Asn Ala Pro Met Thr Ser Val Ala Pro Pro 340 345 350 Pro Ser Val Leu Ser Gln Glu Pro Arg Leu Leu Ile Ser Gly Met Asp 355 360 365 Gln Pro Leu Pro Leu Arg Thr Asp Leu Ile 370 375 <210> 11 <211> 1137 <212> DNA <213> Mus musculus <400> 11 atgccatact ccaacctgca tccagccatc ccacggccca gaggtcaccg ctccaaatat 60 gtagccctca tctttctggt ggccagcctg atgatccttt gggtggcaaa ggatccacca 120 aatcacactc tgaagtacct agcacttcac ctagcctcgc acgaacttgg actactgttg 180 aaaaacctct gctgtctggc tgaagagctg tgccatgtcc agtccaggta ccagggcagc 240 tactggaagg ctgtgcgcgc ctgcctggga tgccccatcc actgtatggc tatgattcta 300 ctatcgtctt atttctattt cctccaaaac actgctgaca tatacctcag ttggatgttt 360 ggccttctgg tcctctataagtccctaagc atgctcctgg gccttcagag cttgactcca 420 gcggaagtct ctgcagtctg tgaagaaaag aagttaaatg ttgcccacgg gctggcctgg 480 tcatactaca ttgggtactt gcggttgatc ttaccagggc tccaggcccg gatccgaatg 540 ttcaatcagc tacataacaa catgctcagt ggtgcaggga gccgaagact gtacatcctc 600 tttccattgg actgtggggt gcctgacaac ctgagtgtag ttgaccccaa cattcgattc 660 cgagatatgc tgccccagca aaacatcgac cgtgctggca tcaagaatcg ggtttattcc 720 aacagcgtct acgagattct ggagaacgga cagccagcag gcgtctgtat cctggagtac 780 gccaccccct tgcagaccct gtttgccatg tcacaggatg ccaaagctgg cttcagtcgg 840 gaggatcggc ttgagcaggc taaactcttc tgccggacac ttgaggaaat cctggaagat 900 gtccccgagt ctcgaaataa ctgccgcctc attgtctacc aagaacccac agacggaaac 960 agtttctcac tgtctcagga ggtgctccgg cacattcgtc aggaagaaaa ggaggaggtt 1020 accatgaatg cccccatgac ctcagtggca cctcctccct ccgtactgtc ccaagagcca 1080 agactcctca tcagtggtat ggatcagcct ctcccactcc gcactgacct catctga 1137 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cgtgctggca tcaaggatcg ggtttac 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gtcaccggtc tgctggggca gtttatc 27 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctgaccgtg ctggcatcaa ggatcgggtt tac 33 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ggtctgctgg ggcagtttat ccagg 25 <210> 16 <211> 24 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 16 caccacatcc actccaggta ccgg 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cagctcctca gccaggctgc agac 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cagacacttg aggacatcct ggcag 25 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcagaagagt ttggcctgct caa 23 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 acctgtattt tcagggcctg gccccagctg agatctctg 39 <210> 21 <211> 44 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 21 cagaattcgc aagcttttaa gtaacctctt ccttttcctc ctgc 44 <210> 22 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139- 342) human WT <400> 22 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile A rg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr < 210> 23 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) human REF mutant <400> 23 Met Gly Ser Ser His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 24 <211> 243 <212> PRT <213> Artificial Sequence <220 > <223> HisAviTEV-hSTING (139-342) human AQ mutant <400> 24 Met Gly Ser Ser His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Ala Pro Ala Glu Ile Ser Ala Val 35 40 45 Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala Arg Ile 65 70 75 80 Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala Val Ser 85 90 95 Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu Pro Gln 115 120 125 Gln Thr Ala Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr Ser Asn Ser 130 135 140 Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys Val Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Tyr Ser 165 170 175 Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Gln Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser Gln Asn 195 200 205 Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu Glu Ly s Glu 225 230 235 240 Glu Val Thr <210> 25 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> HisAviTEV-hSTING (139-342) mouse WT <400> 25 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Gly Leu 1 5 10 15 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Gly Ser 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Leu Thr Pro Ala Glu Val Ser Ala Val 35 40 45 Cys Glu Glu Lys Lys Leu Asn Val Ala His Gly Leu Ala Trp Ser Tyr 50 55 60 Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Gly Leu Gln Ala Arg Ile 65 70 75 80 Arg Met Phe Asn Gln Leu His Asn Asn Met Leu Ser Gly Ala Gly Ser 85 90 95 Arg Arg Leu Tyr Ile Leu Phe Pro Leu Asp Cys Gly Val Pro Asp Asn 100 105 110 Leu Ser Val Val Asp Pro Asn Ile Arg Phe Arg Asp Met Leu Pro Gln 115 120 125 Gln Asn Ile Asp Arg Ala Gly Ile Lys Asn Arg Val Tyr Ser Asn Ser 130 135 140 Val Tyr Glu Ile Leu Glu As n Gly Gln Pro Ala Gly Val Cys Ile Leu 145 150 155 160 Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln Asp Ala 165 170 175 Lys Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys Leu Phe 180 185 190 Cys Arg Thr Leu Glu Glu Ile Leu Glu Asp Val Pro Glu Ser Arg Asn 195 200 205 Asn Cys Arg Leu Ile Val Tyr Gln Glu Pro Thr Asp Gly Asn Ser Phe 210 215 220 Ser Leu Ser Gln Glu Val Leu Arg His Ile Arg Gln Glu Glu Lys Glu 225 230 235 240 Glu Val Thr <210> 26 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Modified LPS Antibody Light Chain <400> 26 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ala Ser Glu Asn Val Gly Asn Ser 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr His Cys Gly Gln Ser Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 27 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Modified LPS Antibody Heavy Chain <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Gly Thr Arg Ser Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Cys 85 90 95 Thr Arg Val Tyr Tyr Asp His Val Gly Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab Light Chain <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Se r Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 449 < 212> PRT <213> Artificial Sequence <220> <223> Pertuzumab Heavy Chain <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly A la Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr V al Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 30 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Modified HER2 Antibody2 Heavy Chain <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 31 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD33 Antibody Light Chain <400> 31 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr 20 25 30 Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 32 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD33 Antibody Heavy Chain <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 33 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Anti-EphA2 Antibody Light Chain <400> 33 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 34 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Anti-EphA2 Antibody Heavy Chain <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 35 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Anti-CDH6 Antibody Light Chain <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Tyr Lys Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Thr Leu Gln Thr Gly Val Pro Ser Ar g Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Ala 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 36 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Anti-CDH6 Antibody Heavy Chain <400> 36 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Phe Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Glu Thr Glu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Tyr Gly Gly Phe Ala Gly Gly Tyr Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp A sn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser V al Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gl y Lys 450

Claims (72)

다음 식 (II) :
Figure pat00635

(식 중, m1 은 1 내지 10 의 범위이고,
Ab 는, 항체 또는 그 항체의 기능성 단편을 나타내고, 그 항체의 당사슬은 리모델링 되어 있어도 되고,
L 은, Ab 와 D 를 연결하는 링커를 나타내고,
Ab 는 그 아미노산 잔기로부터 직접 L 에 결합하거나, Ab 의 당사슬 또는 리모델링 된 당사슬로부터 L 에 결합하고 있어도 되고,
D 는, 다음 식 (I) :
Figure pat00636

(여기서,
L 은, L1 또는 L2 에 포함되는 임의의 -NH2 또는 하이드록시기와 결합하고,
L1 은, 하이드록시기, -NH2, 2-하이드록시아세틸아미노메틸기 및 2-[(2-하이드록시아세틸)아미노]에틸기로 이루어지는 군에서 선택되는 1 내지 3 개의 기에 의해 임의의 위치에서 치환되어 있어도 되는, 다음 식
Figure pat00637

(여기서,
R6 및 R6' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기, -NH2, C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기를 나타내고,
R7 및 R7' 는, 각각 독립적으로, 수소 원자 또는 C1-C6 알킬기를 나타내고, 그 C1-C6 알킬기는, 할로겐 원자 및 옥소기로 이루어지는 군에서 선택되는 1 또는 2 개의 치환기에 의해 치환되어 있어도 되고,
R8 및 R8' 는, 각각 독립적으로, 수소 원자 또는 할로겐 원자를 나타내고,
Z4 는, -CH2-, -NH- 또는 산소 원자를 나타내고,
Z5 는, 질소 원자 또는 -CH= 를 나타낸다)
으로 이루어지는 군에서 선택되는 기를 나타내고,
L2 는, 하기 (i) 또는 (ii) :
(i) L 과 결합할 때, L2 는, -NHR', 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타내고, 여기서, R' 는, 수소 원자, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타내고, 그 C1-C6 알킬기, C2-C6 알케닐기 또는 C2-C6 알키닐기는, 1 내지 6 개의 할로겐 원자로 치환되어도 된다 ; 또는
(ii) L 과 결합하지 않을 때, L2 는, 수소 원자 또는 할로겐 원자를 나타낸다,
에서 선택되는 기를 나타내고,
Q1 및 Q1' 는, 각각 독립적으로, 하이드록시기, 티올기 또는 보라노기 (BH3 -) 를 나타내고,
Q2 및 Q2' 는, 각각 독립적으로, 산소 원자 또는 황 원자를 나타내고,
X1 및 X2 는, 각각 독립적으로, 산소 원자, 황 원자 또는 -CH2- 를 나타내고,
Y1 및 Y2 는, 산소 원자 또는 -CH2- 를 나타내고,
X3 및 X4 는, 하기 (iii) 또는 (iv) :
(iii) Y1 이 산소 원자일 때, X3-X4 는, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는
(iv) Y1 이 -CH2- 일 때, X3-X4 는, -O-CH2- 를 나타낸다,
에서 선택되는 기를 나타내고,
X5 및 X6 은, 하기 (v) 또는 (vi) :
(v) Y2 가 산소 원자일 때, X5-X6 은, -CH2-O-, -CH2-S-, -CH2-CH2- 또는 -CH2-CF2- 를 나타낸다 ; 또는
(vi) Y2 가 -CH2- 일 때, X5-X6 은, -O-CH2- 를 나타낸다,
에서 선택되는 기를 나타내고,
R1, R2 및 R3 은, 각각 독립적으로, 수소 원자, 할로겐 원자, -OR', -OC(=O)R', -N3, -NHR', -NR'R'' 또는 -NHC(=O)R' (여기서, R' 는, 앞에 정의한 바와 같고, R'' 는, C1-C6 알킬기, C2-C6 알케닐기, C2-C6 알키닐기 또는 C3-C6 시클로알킬기를 나타낸다) 를 나타내고,
W1 은, 질소 원자, 산소 원자, 황 원자 또는 -CH- 를 나타내고,
W2 는, 질소 원자 또는 -CH= 를 나타내고,
R4 는, 수소 원자, 할로겐 원자 또는 -NH2 를 나타내고,
R5 는, 하기 (vii) 내지 (x) :
(vii) W1 이 질소 원자일 때, R5 는 수소 원자, C1-C6 알킬기, 하이드록시 C1-C6 알킬기 또는 아미노 C1-C6 알킬기를 나타낸다 ;
(viii) W1 이 산소 원자일 때, R5 는 존재하지 않고 ;
(ix) W1 이 황 원자일 때, R5 는 존재하지 않고 ; 또는
(x) W1 이 -CH- 일 때, R5 는 수소 원자, 할로겐 원자, 하이드록시기, -NH2 또는 C1-C6 알킬기를 나타낸다,
에서 선택되는 기를 나타내고,
Z1-Z2-Z3 은, 하나가 되어, -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타내고, X 는, 할로겐 원자를 나타낸다) 또는 하기 식 중 어느 것으로 나타내는 기
Figure pat00638

(여기서, 아스테리스크는, W1 에 결합하고 있는 것을 나타내고, 파선은, =C- 의 탄소 원자에 결합하고 있는 것을 나타낸다) 에서 선택되는 기를 나타낸다)
로 나타내는 화합물을 나타낸다)
로 나타내고, 단, 다음 식 (II) :
Figure pat00639

(식 중, m1 은 3 내지 5 의 범위이고,
Ab 는, 항HER2 항체를 나타내고, 그 항체의 당사슬은 리모델링 되어 있고,
L 은, Ab 와 D 를 연결하는 링커를 나타내고,
링커 L 이 -Lb-La-Lp-Lc-* 로 나타내고,
식 중, 아스테리스크는, 약물 D 와 결합하고 있는 것을 나타내고,
Lp 는, -GGFG- 이고,
La 는, -C(=O)-CH2CH2-C(=O)- 를 나타내고,
Lb 는, 다음 식 :
Figure pat00640

를 나타내고, 상기에서 나타내는 Lb 의 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 리모델링된 당사슬과 결합하고 있는 것을 나타내고, 및
Lc 는, -NH-CH2- 를 나타내고,
Ab 는, 항체의 Asn297 에 결합하는 당사슬 (N297-(Fuc)SG) 로부터 L 에 결합하고 있고, 그리고,
D 는, 다음 식 (I) :
Figure pat00641

(여기서, 아스테리스크는, L 과 결합하고 있는 것을 나타낸다) 로 나타내는 화합물을 나타낸다) 로 나타내는 항체 약물 콘쥬게이트는 제외하는, 항체 약물 콘쥬게이트.Equation (II):
Figure pat00635

(In the formula, m 1 is in the range of 1 to 10,
Ab represents an antibody or a functional fragment of the antibody, the sugar chain of the antibody may be remodeled,
L represents a linker linking Ab and D,
Ab may bind to L directly from its amino acid residue, or may bind to L from an Ab sugar chain or a remodeled sugar chain;
D is the following formula (I):
Figure pat00636

(here,
L is bonded to any -NH 2 or hydroxyl group included in L 1 or L 2 ;
L 1 is substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxyl group, -NH 2 , a 2-hydroxyacetylaminomethyl group and a 2-[(2-hydroxyacetyl)amino]ethyl group The following expression, which may be
Figure pat00637

(here,
R 6 and R 6' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 , a C1-C6 alkyl group, a C2-C6 alkenyl group or a C2-C6 alkynyl group;
R 7 and R 7' each independently represent a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group may be substituted with 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group; ,
R 8 and R 8' each independently represent a hydrogen atom or a halogen atom;
Z 4 represents -CH 2 -, -NH- or an oxygen atom;
Z 5 represents a nitrogen atom or -CH=)
represents a group selected from the group consisting of
L 2 is the following (i) or (ii):
(i) When combined with L, L 2 represents -NHR', a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group, wherein R' is a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alky represents a nyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group may be substituted with 1 to 6 halogen atoms; or
(ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom;
represents a group selected from
Q 1 and Q 1' each independently represent a hydroxy group, a thiol group or a borano group (BH 3 - );
Q 2 and Q 2' each independently represents an oxygen atom or a sulfur atom;
X 1 and X 2 each independently represent an oxygen atom, a sulfur atom or -CH 2 -;
Y 1 and Y 2 represent an oxygen atom or -CH 2 -;
X 3 and X 4 are the following (iii) or (iv):
(iii) when Y 1 is an oxygen atom, X 3 -X 4 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or
(iv) When Y 1 is -CH 2 -, X 3 -X 4 represents -O-CH 2 -;
represents a group selected from
X 5 and X 6 are (v) or (vi):
(v) when Y 2 is an oxygen atom, X 5 -X 6 represents -CH 2 -O-, -CH 2 -S-, -CH 2 -CH 2 - or -CH 2 -CF 2 -; or
(vi) When Y 2 is -CH 2 -, X 5 -X 6 represents -O-CH 2 -;
represents a group selected from
R 1 , R 2 and R 3 are each independently a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N 3 , -NHR', -NR'R'' or -NHC (=O)R' (where R' is as defined above, and R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group) ,
W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom or -CH-;
W 2 represents a nitrogen atom or -CH=;
R 4 represents a hydrogen atom, a halogen atom or -NH 2 ;
R 5 is the following (vii) to (x):
(vii) When W 1 is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group;
(viii) when W 1 is an oxygen atom, R 5 is absent;
(ix) when W 1 is a sulfur atom, R 5 is absent; or
(x) When W 1 is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH 2 or a C1-C6 alkyl group;
represents a group selected from
Z 1 -Z 2 -Z 3 is one, -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -R'''-, -CH=CH-CH 2 -, -CH= CX-CH 2 -, -CX=CH-CH 2 -, -CX=CX-CH 2 -, -C(=O)-CH 2 -CH 2 -, -CH 2 -CH 2 -C(=O) -, -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )- (where R''' is -O- or -CH 2 -CH 2 - and X represents a halogen atom) or a group represented by any of the following formulas
Figure pat00638

(Here, the asterisk indicates bonded to W 1 , and the broken line indicates bonded to a carbon atom of =C-).)
represents the compound represented by)
Represented by, provided that the following formula (II):
Figure pat00639

(In the formula, m 1 is in the range of 3 to 5,
Ab represents an anti-HER2 antibody, and the sugar chain of the antibody is remodeled;
L represents a linker linking Ab and D,
Linker L is represented by -Lb-La-Lp-Lc-*,
In the formula, Asterisk represents binding to drug D,
Lp is -GGFG-;
La represents -C(=O)-CH 2 CH 2 -C(=O)-;
Lb is the following expression:
Figure pat00640

In the structural formula of Lb shown above, the asterisk indicates binding to La, the broken line indicates binding to the remodeled sugar chain of Ab, and
Lc represents -NH-CH 2 -;
Ab is bound to L from the sugar chain (N297-(Fuc)SG) that binds to Asn297 of the antibody, and
D is the following formula (I):
Figure pat00641

Antibody drug conjugates, excluding antibody drug conjugates represented by (here, Asterisk represents a compound represented by L). 제 1 항에 있어서,
W1 이 질소 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein W 1 is a nitrogen atom. 제 2 항에 있어서,
W1 이 질소 원자이고, R5 가 수소 원자인, 항체 약물 콘쥬게이트.According to claim 2,
An antibody drug conjugate wherein W 1 is a nitrogen atom and R 5 is a hydrogen atom. 제 1 항에 있어서,
W1 이 산소 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein W 1 is an oxygen atom. 제 1 항에 있어서,
W1 이 황 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein W 1 is a sulfur atom. 제 1 항에 있어서,
W1 이 -CH- 인, 항체 약물 콘쥬게이트.According to claim 1,
and wherein W 1 is -CH-. 제 6 항에 있어서,
W1 이 -CH- 이고, R5 가 수소 원자인, 항체 약물 콘쥬게이트.According to claim 6,
An antibody drug conjugate wherein W 1 is -CH- and R 5 is a hydrogen atom. 제 1 항에 있어서,
Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-CH2- 또는 -CH=CH-CH2- 인, 항체 약물 콘쥬게이트.According to claim 1,
Z 1 , Z 2 and Z 3 taken together are -CH 2 -CH 2 -CH 2 - or -CH=CH-CH 2 -, an antibody drug conjugate. 제 1 항에 있어서,
Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH(CH3)-CH2- 또는 -CH2-CH2-CH(CH3)- 인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein Z 1 , Z 2 and Z 3 are, taken together, -CH 2 -CH(CH 3 )-CH 2 - or -CH 2 -CH 2 -CH(CH 3 )-. 제 1 항에 있어서,
Z1, Z2 및 Z3 이, 하나가 되어, -CH2-CH2-R'''- (여기서, R''' 는, -O- 또는 -CH2-CH2- 를 나타낸다) 인, 항체 약물 콘쥬게이트.According to claim 1,
Z 1 , Z 2 and Z 3 are, together, -CH 2 -CH 2 -R'''- (where R''' represents -O- or -CH 2 -CH 2 -). , Antibody drug conjugates. 제 1 항에 있어서,
W2 가 -CH= 인, 항체 약물 콘쥬게이트.According to claim 1,
and wherein W 2 is -CH=. 제 1 항에 있어서,
W2 가 질소 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which W 2 is a nitrogen atom. 제 1 항에 있어서,
R4 가, 수소 원자를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which R 4 represents a hydrogen atom. 제 1 항에 있어서,
R4 가, 불소 원자를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which R 4 represents a fluorine atom. 제 1 항에 있어서,
L1 중의 R8 및 R8' 가, 각각 독립적으로, 수소 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which R 8 and R 8' in L 1 are each independently a hydrogen atom. 제 1 항에 있어서,
L1 이, 다음 식으로 이루어지는 군에서 선택되는 기
Figure pat00642

(여기서, R9 및 R9' 는, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고,
R10 은, 하이드록시기, -NH2, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, 하이드록시 C1-C3 알킬기 또는 아미노 C1-C3 알킬기를 나타내고,
R11 및 R11' 는, 각각 독립적으로, 수소 원자, 불소 원자 혹은 메틸기를 나타내거나, 또는, R11 및 R11' 가 결합하여 시클로프로판을 나타내고,
Z4 는, -CH2-, -NH- 또는 산소 원자를 나타낸다)
인, 항체 약물 콘쥬게이트.According to claim 1,
L 1 is a group selected from the group consisting of the following formula
Figure pat00642

(Where, R 9 and R 9' represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ,
R 10 is a hydroxy group, -NH 2 , -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH, -CH 2 CH 2 NHC(=O)CH 2 OH, hydroxy represents a C1-C3 alkyl group or an amino C1-C3 alkyl group;
R 11 and R 11' each independently represent a hydrogen atom, a fluorine atom or a methyl group, or R 11 and R 11' combine to represent cyclopropane;
Z 4 represents -CH 2 -, -NH- or an oxygen atom)
phosphorus, antibody drug conjugates. 제 1 항에 있어서,
L1 이, 다음 식으로 이루어지는 군에서 선택되는 기
Figure pat00643

(여기서, R13 및 R13' 는, 각각 독립적으로, 수소 원자, 하이드록시기 또는 -NH2 를 나타내고,
R12 는, 하이드록시기, -NH2, -CH2OH, -NHC(=O)CH2OH, -CH2NHC(=O)CH2OH 또는 -CH2CH2NHC(=O)CH2OH 를 나타내고,
Z4 는, 앞에 정의한 바와 같다)
인, 항체 약물 콘쥬게이트.According to claim 1,
L 1 is a group selected from the group consisting of the following formula
Figure pat00643

(Where, R 13 and R 13' each independently represent a hydrogen atom, a hydroxyl group, or -NH 2 ,
R 12 is a hydroxy group, -NH 2 , -CH 2 OH, -NHC(=O)CH 2 OH, -CH 2 NHC(=O)CH 2 OH or -CH 2 CH 2 NHC(=O)CH 2 OH,
Z 4 is as defined above)
phosphorus, antibody drug conjugates. 제 1 항에 있어서,
L1 이, 다음 식으로 이루어지는 군에서 선택되는 기
Figure pat00644

(여기서, R14 는, 수소 원자 또는 -NH2 를 나타내고,
R15 는, 수소 원자 또는 -C(=O)CH2OH 를 나타내고,
R16 은, 하이드록시기, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 또는 -CH2CH2NH2 를 나타낸다)
인, 항체 약물 콘쥬게이트.According to claim 1,
L 1 is a group selected from the group consisting of the following formula
Figure pat00644

(Here, R 14 represents a hydrogen atom or -NH 2 ,
R 15 represents a hydrogen atom or -C(=O)CH 2 OH;
R 16 represents a hydroxy group, -NH 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 NH 2 or -CH 2 CH 2 NH 2 )
phosphorus, antibody drug conjugates. 제 1 항에 있어서,
L2 가, L 과 결합하고, -NH2, -CH2NH2 또는 -CH2OH 를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
L 2 is combined with L and represents -NH 2 , -CH 2 NH 2 or -CH 2 OH. 제 1 항에 있어서,
L2 가, L 과 결합하지 않고, 수소 원자 또는 불소 원자를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which L 2 is not bonded to L and represents a hydrogen atom or a fluorine atom. 제 1 항에 있어서,
Q1 및 Q1' 가, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which Q 1 and Q 1' each independently represent a hydroxy group or a thiol group. 제 1 항에 있어서,
X1 및 X2 가, 산소 원자를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein X 1 and X 2 represent oxygen atoms. 제 1 항에 있어서,
Y1 및 Y2 가, 산소 원자를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein Y 1 and Y 2 represent oxygen atoms. 제 1 항에 있어서,
X3 및 X4 가, -CH2-O- 를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate wherein X 3 and X 4 represent -CH 2 -O-. 제 1 항에 있어서,
X5 및 X6 이, -CH2-O- 를 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
The antibody drug conjugate, wherein X 5 and X 6 represent -CH 2 -O-. 제 1 항에 있어서,
R1, R2 및 R3 이, 각각 독립적으로, 수소 원자, 하이드록시기 또는 불소 원자인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which R 1 , R 2 and R 3 are each independently a hydrogen atom, a hydroxyl group or a fluorine atom. 제 1 항에 있어서,
D 가, 이하의 2 식 :
Figure pat00645

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,
R17, R17', R18 및 R18' 는, 각각 독립적으로, 수소 원자, 할로겐 원자, 하이드록시기 또는 -NH2 를 나타내고,
W3 은, -NH-, 산소 원자, 황 원자 또는 -CH2- 를 나타내고,
W4 는, -CH= 또는 질소 원자를 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
D is the following 2 equations:
Figure pat00645

(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,
R 17 , R 17' , R 18 and R 18' each independently represent a hydrogen atom, a halogen atom, a hydroxyl group or -NH 2 ;
W 3 represents -NH-, an oxygen atom, a sulfur atom or -CH 2 -;
W 4 represents -CH= or a nitrogen atom)
An antibody drug conjugate represented by any of the above. 제 27 항에 있어서,
D 가, 이하의 2 식 :
Figure pat00646

(여기서, L1, Q1, Q1', Q2, Q2', R17, R17', R18 및 R18' 는, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.28. The method of claim 27,
D is the following 2 equations:
Figure pat00646

(Where, L 1 , Q 1 , Q 1' , Q 2 , Q 2' , R 17 , R 17' , R 18 and R 18' are as defined above)
An antibody drug conjugate represented by any of the above. 제 27 항에 있어서,
D 가, 이하의 8 식 :
Figure pat00647

(여기서, L1, Q1, Q1', Q2 및 Q2' 는, 앞에 정의한 바와 같고,
R19, R19', R20 및 R20' 는, 각각 독립적으로, 수소 원자 또는 불소 원자를 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.28. The method of claim 27,
D is, the following 8 expression:
Figure pat00647

(Where L 1 , Q 1 , Q 1 ' , Q 2 and Q 2' are as defined above,
R 19 , R 19' , R 20 and R 20' each independently represent a hydrogen atom or a fluorine atom)
An antibody drug conjugate represented by any of the above. 제 27 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00648

(여기서, L1 은, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.28. The method of claim 27,
D, the following 4 equations:
Figure pat00648

(Here, L 1 is as defined above)
An antibody drug conjugate represented by any of the above. 제 27 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00649

(여기서, L1 은, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.28. The method of claim 27,
D, the following 4 equations:
Figure pat00649

(Here, L 1 is as defined above)
An antibody drug conjugate represented by any of the above. 제 27 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00650

(여기서, L1 은, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.28. The method of claim 27,
D, the following 4 equations:
Figure pat00650

(Here, L 1 is as defined above)
An antibody drug conjugate represented by any of the above. 제 1 항에 있어서,
D 가, 다음 식 :
Figure pat00651

(여기서, L1 은, 앞에 정의한 바와 같고,
Q3 및 Q3' 는, 각각 독립적으로, 하이드록시기 또는 티올기를 나타내고,
R21 및 R22 는, 각각 독립적으로, 하이드록시기 또는 불소 원자를 나타내고,
W5 는, -NH- 또는 황 원자를 나타낸다)
으로 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
D, the following expression:
Figure pat00651

(Where L 1 is as defined above,
Q 3 and Q 3' each independently represent a hydroxy group or a thiol group;
R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom;
W 5 represents -NH- or a sulfur atom)
An antibody drug conjugate, represented by . 제 33 항에 있어서,
D 가, 이하의 2 식 :
Figure pat00652

(여기서, L1, Q3 및 Q3', W5 는, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.34. The method of claim 33,
D is the following 2 equations:
Figure pat00652

(Here, L 1 , Q 3 and Q 3 ' , W 5 are as defined above)
An antibody drug conjugate represented by any of the above. 제 1 항에 있어서,
L1 이, 이하의 4 식 :
Figure pat00653

중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
L 1 has the following 4 expressions:
Figure pat00653

An antibody drug conjugate represented by any of the above. 제 1 항에 있어서,
L1 이, 이하의 4 식 :
Figure pat00654

(식 중, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.According to claim 1,
L 1 has the following 4 expressions:
Figure pat00654

(In the formula, asterisk indicates bonding with L)
An antibody drug conjugate represented by any of the above. 제 33 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00655

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타내고, Q3, Q3' 및 W5 는, 앞에 정의한 바와 같다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.34. The method of claim 33,
D, the following 4 equations:
Figure pat00655

(Here, the asterisk indicates that it is bonded to L, and Q 3 , Q 3' and W 5 are as defined above)
An antibody drug conjugate represented by any of the above. 제 33 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00656

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.34. The method of claim 33,
D, the following 4 equations:
Figure pat00656

(Here, the asterisk indicates that it is bonded to L)
An antibody drug conjugate represented by any of the following. 제 33 항에 있어서,
D 가, 이하의 3 식 :
Figure pat00657

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.34. The method of claim 33,
D is the following 3 expressions:
Figure pat00657

(Here, the asterisk indicates that it is bonded to L)
An antibody drug conjugate represented by any of the above. 제 33 항에 있어서,
D 가, 이하의 4 식 :
Figure pat00658

(여기서, 아스테리스크는 L 과 결합하고 있는 것을 나타낸다)
중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.34. The method of claim 33,
D, the following 4 equations:
Figure pat00658

(Here, the asterisk indicates that it is bonded to L)
An antibody drug conjugate represented by any of the above. 제 1 항에 있어서,
링커 L 이, -Lb-La-Lp-Lc-* 로 나타내고,
식 중, 아스테리스크는, 약물 D 와 결합하고 있는 것을 나타내고,
Lp 는, 표적 세포에 있어서 절단 가능한 아미노산 서열로 이루어지는 링커를 나타내거나 또는 존재하지 않고,
La 는, 이하의 군에서 선택되는 어느 1 개를 나타내고,
-C(=O)-(CH2CH2)n2-C(=O)-,
-C(=O)-(CH2CH2)n2-CH2-C(=O)-,
-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-C(=O)-,
-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2)n3-CH2-C(=O)-,
-C(=O)-(CH2CH2)n2-C(=O)-NH-(CH2CH2O)n3-CH2-C(=O)-,
-(CH2)n4-O-C(=O)-, 및,
-(CH2)n9-C(=O)-
(여기서, n2 는 1 ∼ 3 의 정수를 나타내고, n3 은 1 ∼ 5 의 정수를 나타내고, n4 는 0 ∼ 2 의 정수를 나타내고, n9 는 2 ∼ 7 의 정수를 나타낸다),
Lb 는, La 와 Ab 의 당사슬 또는 리모델링 된 당사슬을 결합하는 스페이서 또는 La 와 Ab 의 시스테인 잔기를 결합하는 스페이서를 나타내고, 그리고,
Lc 는, -NH-CH2-, -페닐기-CH2-O(C=O)- 또는 -NH-헤테로아릴기-CH2-O(C=O)- 를 나타내거나 또는 존재하지 않는, 항체 약물 콘쥬게이트.According to claim 1,
Linker L is represented by -Lb-La-Lp-Lc-*,
In the formula, Asterisk represents binding to drug D,
Lp represents or does not exist a linker consisting of an amino acid sequence cleavable in the target cell;
La represents any one selected from the following groups,
-C(=O)-(CH 2 CH 2 )n 2 -C(=O)-,
-C(=O)-(CH 2 CH 2 )n 2 -CH 2 -C(=O)-,
-C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-;
-C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -CH 2 -C(=O)-,
-C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-,
-(CH 2 )n 4 -OC(=O)-, and
-(CH 2 )n 9 -C(=O)-
(Where n 2 represents an integer of 1 to 3, n 3 represents an integer of 1 to 5, n 4 represents an integer of 0 to 2, and n 9 represents an integer of 2 to 7),
Lb represents a spacer that binds the sugar chains of La and Ab or the remodeled sugar chains or a spacer that binds the cysteine residues of La and Ab, and
Lc represents -NH-CH 2 -, -phenyl group-CH 2 -O(C=O)-, or -NH-heteroaryl group-CH 2 -O(C=O)-, or is absent; antibody drug conjugates. 제 41 항에 있어서,
Lc 가 존재하지 않는, 항체 약물 콘쥬게이트.42. The method of claim 41,
An antibody drug conjugate in which Lc is not present. 제 41 항에 있어서,
Lc 가 -NH-CH2- 인, 항체 약물 콘쥬게이트.42. The method of claim 41,
and Lc is -NH-CH 2 -. 제 41 항에 있어서,
Lp 가, 이하 :
-GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit- 및 -ICit- 로 이루어지는 군에서 선택되는 어느 1 개를 나타내는, 항체 약물 콘쥬게이트.42. The method of claim 41,
Lp is, or less:
-GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM An antibody drug conjugate which represents any one selected from the group consisting of -, -FM-, -GGLM-, -LM-, -GGICit- and -ICit-. 제 44 항에 있어서,
Lp 가, -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit- 또는 -FCit- 중 어느 1 개인, 항체 약물 콘쥬게이트.45. The method of claim 44,
An antibody drug conjugate in which Lp is any one of -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, or -FCit-. 제 41 항에 있어서,
Lp 가, -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ- 또는 -GGPP- 중 어느 1 개인, 항체 약물 콘쥬게이트.42. The method of claim 41,
Lp is -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-, an antibody drug conjugate gate. 제 46 항에 있어서,
Lp 가, -GGFG- 또는 -GGPI- 인, 항체 약물 콘쥬게이트.47. The method of claim 46,
An antibody drug conjugate wherein Lp is -GGFG- or -GGPI-. 제 41 항에 있어서,
La 가, 이하 :
-C(=O)-CH2CH2-C(=O)-,
-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-,
-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, 및
-(CH2)5-C(=O)-
로 이루어지는 군에서 선택되는 어느 1 개를 나타내는, 항체 약물 콘쥬게이트.42. The method of claim 41,
La, below:
-C(=O)-CH 2 CH 2 -C(=O)-,
-C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 3 -CH 2 -C(=O)-,
-C(=0)-CH 2 CH 2 -C(=0)-NH-(CH 2 CH 2 O) 4 -CH 2 -C(=0)-, and
-(CH 2 ) 5 -C(=O)-
Antibody drug conjugate, which represents any one selected from the group consisting of. 제 41 항에 있어서,
Lb 가, 다음 식
Figure pat00659

Figure pat00660

또는,
Figure pat00661

(상기에서 나타내는 Lb 의 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 당사슬 또는 리모델링 된 당사슬과 결합하고 있는 것을 나타낸다) 중 어느 것으로 나타내는, 항체 약물 콘쥬게이트.42. The method of claim 41,
Lb is, the following expression
Figure pat00659

Figure pat00660

or,
Figure pat00661

(In the structural formula of Lb shown above, the asterisk indicates binding to La, and the broken line indicates binding to Ab sugar chain or remodeled sugar chain). 제 41 항에 있어서,
Lb 가, -(숙신이미드-3-일-N)- 이고,
여기서, -(숙신이미드-3-일-N)- 는, 이하의 구조식 :
Figure pat00662

을 나타내고,
여기서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 항체의 시스테인 잔기의 측사슬과 티오에테르를 형성하여 결합하고 있는 것을 나타내는, 항체 약물 콘쥬게이트.42. The method of claim 41,
Lb is -(succinimide-3-yl-N)-,
Here, -(succinimide-3-yl-N)- is the following structural formula:
Figure pat00662

represents,
Here, the asterisk indicates binding to La, and the dashed line indicates binding by forming a thioether with the side chain of the cysteine residue of the antibody. 제 41 항에 있어서,
링커 L 이, -Lb-La-Lp-Lc-* 로 나타내고,
식 중, 아스테리스크는, 약물 D 와 결합하고 있는 것을 나타내고,
Lp 가, -GGFG-, 또는 -GGPI- 이고,
La 가, -C(=O)-CH2CH2-C(=O)- 를 나타내고,
Lb 가, 다음 식
Figure pat00663

(상기에서 나타내는 Lb 의 구조식에 있어서, 아스테리스크는 La 와 결합하고 있는 것을 나타내고, 파선은 Ab 의 당사슬 또는 리모델링 된 당사슬과 결합하고 있는 것을 나타낸다) 을 나타내고,
Lc 가, -NH-CH2- 를 나타내는, 항체 약물 콘쥬게이트.42. The method of claim 41,
Linker L is represented by -Lb-La-Lp-Lc-*,
In the formula, Asterisk represents binding to drug D,
Lp is -GGFG- or -GGPI-;
La represents -C(=O)-CH 2 CH 2 -C(=O)-;
Lb is, the following expression
Figure pat00663

(In the structural formula of Lb shown above, the asterisk indicates that it is bonded to La, and the broken line indicates that it is bonded to the sugar chain of Ab or the remodeled sugar chain),
wherein Lc represents -NH-CH 2 -. 제 1 항에 있어서,
항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 평균 약물 결합수가, 1 내지 10 개의 범위인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which the average number of drug bonds per antibody molecule in the antibody drug conjugate is in the range of 1 to 10. 제 52 항에 있어서,
항체 약물 콘쥬게이트에 있어서의 항체 1 분자당의 평균 약물 결합수가, 1 내지 5 개의 범위인, 항체 약물 콘쥬게이트.53. The method of claim 52,
An antibody drug conjugate in which the average number of drug bonds per antibody molecule in the antibody drug conjugate is in the range of 1 to 5. 제 1 항에 있어서,
항체가, 항체의 Asn297 에 결합하는 당사슬 (N297 당사슬) 로부터 L 에 결합하고 있는, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate in which an antibody is bound to L from a sugar chain (N297 sugar chain) bound to Asn297 of the antibody. 제 54 항에 있어서,
N297 당사슬이, 리모델링 된 당사슬인, 항체 약물 콘쥬게이트.55. The method of claim 54,
An antibody drug conjugate wherein the N297 sugar chain is a remodeled sugar chain. 제 54 항에 있어서,
N297 당사슬이, N297-(Fuc)MSG1 또는 N297-(Fuc)SG 인, 항체 약물 콘쥬게이트.55. The method of claim 54,
An antibody drug conjugate wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG. 제 1 항에 있어서,
항체 또는 항체의 항원 결합 단편이, 항HER2 항체, 항HER3 항체, 항DLL3 항체, 항FAP 항체, 항CDH11 항체, 항CDH6 항체, 항A33 항체, 항CanAg 항체, 항CD19 항체, 항CD20 항체, 항CD22 항체, 항CD30 항체, 항CD33 항체, 항CD56 항체, 항CD70 항체, 항CD98 항체, 항TROP2 항체, 항CEA 항체, 항Cripto 항체, 항EphA2 항체, 항G250 항체, 항MUC1 항체, 항GPNMB 항체, 항Integrin 항체, 항PSMA 항체, 항Tenascin-C 항체, 항SLC44A4 항체, 항Mesothelin 항체, 항ENPP3 항체, 항CD47 항체, 항EGFR 항체, 항GPR20 항체 또는 항DR5 항체 또는 항체의 항원 결합 단편인, 항체 약물 콘쥬게이트.According to claim 1,
Antibodies or antigen binding fragments of antibodies, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-CDH6 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD20 antibody Anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody , Anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody or anti-DR5 antibody or an antigen-binding fragment of an antibody, Antibody drug conjugates. 제 1 항에 있어서,
항체 또는 항체의 항원 결합 단편이, IgG1, IgG2, IgG3 또는 IgG4 인 IgG, IgE, IgM, IgD, IgA1 또는 IgA2 인 IgA 및 IgY 로 이루어지는 군에서 선택되는 어느 것인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of IgA and IgY, which are IgG1, IgG2, IgG3, or IgG4, IgE, IgM, IgD, IgA1, or IgA2. 제 58 항에 있어서,
항체 또는 항체의 항원 결합 단편이 IgG1 인, 항체 약물 콘쥬게이트.59. The method of claim 58,
An antibody drug conjugate wherein the antibody or antigen-binding fragment of the antibody is IgG1. 제 59 항에 있어서,
정상 영역의 아미노산 잔기의 일부가 치환된, 항체 약물 콘쥬게이트.The method of claim 59,
An antibody drug conjugate in which a portion of amino acid residues in the constant region are substituted. 제 60 항에 있어서,
치환이, EU 인덱스에 의해 특정되는 234 위치 및 235 위치의 류신의 알라닌으로의 치환을 포함하는, 항체 약물 콘쥬게이트.61. The method of claim 60,
The antibody drug conjugate, wherein the substitution comprises substitution of alanine for leucine at positions 234 and 235 specified by the EU index. 제 1 항 있어서,
항체 또는 항체의 항원 결합 단편이 유전자 공학에 의해 개변된 항체 유전자를 사용하여 숙주 세포에서 산생된 단백질인, 항체 약물 콘쥬게이트.According to claim 1,
An antibody drug conjugate, wherein the antibody or antigen-binding fragment of the antibody is a protein produced in a host cell using an antibody gene modified by genetic engineering. 제 57 항에 있어서,
항체 또는 항체의 항원 결합 단편이, 항HER2 항체 또는 항체의 항원 결합 단편인, 항체 약물 콘쥬게이트.58. The method of claim 57,
An antibody drug conjugate wherein the antibody or antigen-binding fragment of an antibody is an anti-HER2 antibody or an antigen-binding fragment of an antibody. 제 63 항에 있어서,
항체 또는 항체의 항원 결합 단편이, 하기 (i) 내지 (iii) 중 어느 하나인 것인, 항체 약물 콘쥬게이트.
(i) 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 2 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체 또는 항체의 항원 결합 단편, 또는 서열 번호 1 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 3 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체 또는 항체의 항원 결합 단편 ;
(ii) 정상 영역의 아미노산 잔기의 일부가 치환된, (i) 에서 정의된 항체 또는 항체의 항원 결합 단편 ; 및
(iii) (i) 에서 정의된 항체 또는 항체의 항원 결합 단편의 유전자 (b) 로부터 유전자 공학에 의해 개변된 유전자 (a) 를 사용하여 숙주 세포에서 산생된 단백질.64. The method of claim 63,
An antibody drug conjugate, wherein the antibody or antigen-binding fragment of the antibody is any one of the following (i) to (iii).
(i) an antibody or antigen-binding fragment of an antibody comprising a light chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2, or a light chain consisting of the amino acid sequence shown in SEQ ID NO: 1, and an antibody or antigen-binding fragment of an antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 3;
(ii) an antibody or antigen-binding fragment of an antibody as defined in (i) in which a portion of the amino acid residues of the constant region are substituted; and
(iii) A protein produced in a host cell using a gene (a) modified by genetic engineering from a gene (b) of the antibody or antigen-binding fragment of the antibody defined in (i). 제 63 항에 있어서,
항체 또는 항체의 항원 결합 단편이, 하기 (i) 내지 (iii) 중 어느 하나인 것인, 항체 약물 콘쥬게이트.
(i) 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 29 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체 또는 항체의 항원 결합 단편, 또는 서열 번호 28 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 30 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체 또는 항체의 항원 결합 단편 ;
(ii) 정상 영역의 아미노산 잔기의 일부가 치환된, (i) 에서 정의된 항체 또는 항체의 항원 결합 단편 ; 및
(iii) (i) 에서 정의된 항체 또는 항체의 항원 결합 단편의 유전자 (b) 로부터 유전자 공학에 의해 개변된 유전자 (a) 를 사용하여 숙주 세포에서 산생된 단백질.64. The method of claim 63,
An antibody drug conjugate, wherein the antibody or antigen-binding fragment of the antibody is any one of the following (i) to (iii).
(i) an antibody or antigen-binding fragment of an antibody comprising a light chain consisting of the amino acid sequence shown in SEQ ID NO: 28 and a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 29, or a light chain consisting of the amino acid sequence shown in SEQ ID NO: 28, and an antibody or antigen-binding fragment of an antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30;
(ii) an antibody or antigen-binding fragment of an antibody as defined in (i) in which a portion of the amino acid residues of the constant region are substituted; and
(iii) A protein produced in a host cell using a gene (a) modified by genetic engineering from a gene (b) of the antibody or antigen-binding fragment of the antibody defined in (i). 제 64 항에 있어서,
치환이, EU 인덱스에 의해 특정되는 234 위치 및 235 위치의 류신의 알라닌으로의 치환을 포함하는, 항체 약물 콘쥬게이트.65. The method of claim 64,
The antibody drug conjugate, wherein the substitution comprises substitution of alanine for leucine at positions 234 and 235 specified by the EU index. 제 63 항에 있어서,
항체 또는 항체의 항원 결합 단편의 중사슬 카르복실 말단에 있어서 1 또는 2 개의 아미노산이 결실되어 있는, 항체 약물 콘쥬게이트.64. The method of claim 63,
An antibody drug conjugate in which one or two amino acids are deleted from the heavy chain carboxyl terminus of an antibody or an antigen-binding fragment of an antibody. 제 57 항에 있어서,
항체가, 서열 번호 31 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 32 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 서열 번호 33 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 34 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체, 또는, 서열 번호 35 에 기재된 아미노산 서열로 이루어지는 경사슬 및 서열 번호 36 에 기재된 아미노산 서열로 이루어지는 중사슬을 포함하는 항체인, 항체 약물 콘쥬게이트.58. The method of claim 57,
An antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a light chain comprising the amino acid sequence of SEQ ID NO: 33, and an amino acid sequence of SEQ ID NO: 34 An antibody comprising a heavy chain composed of, or an antibody drug conjugate comprising a light chain composed of the amino acid sequence shown in SEQ ID NO: 35 and a heavy chain composed of the amino acid sequence shown in SEQ ID NO: 36. 제 1 항 내지 제 68 항 중 어느 한 항에 기재된 항체 약물 콘쥬게이트로 이루어지는 군에서 선택되는 어느 것을 함유하는, STING 아고니스트.A STING agonist containing any one selected from the group consisting of the antibody drug conjugate according to any one of claims 1 to 68. 제 1 항 내지 제 68 항 중 어느 한 항에 기재된 항체 약물 콘쥬게이트로 이루어지는 군에서 선택되는 어느 것을 함유하는, 의약 조성물.A pharmaceutical composition containing any one selected from the group consisting of the antibody drug conjugate according to any one of claims 1 to 68. 제 1 항 내지 제 68 항 중 어느 한 항에 기재된 항체 약물 콘쥬게이트로 이루어지는 군에서 선택되는 어느 것을 함유하는, 항종양제.An antitumor agent containing any one selected from the group consisting of the antibody drug conjugate according to any one of claims 1 to 68. 제 71 항에 있어서,
종양이, 폐암, 신장암, 요로 상피암, 대장암, 전립선암, 다형 신경교아종, 난소암, 췌암, 유방암, 멜라노마, 간암, 방광암, 위암, 식도암, 자궁체암, 정소암, 자궁경암, 태반 융모암, 다형 신경교아종, 뇌종양, 두경부암, 갑상선암, 중피종, 소화관 간질 종양 (GIST), 담낭암, 담관암, 부신암, 유극세포암, 백혈병, 악성 림프종, 형질 세포종, 골수종 또는 육종인, 항종양제.72. The method of claim 71,
Tumors, lung cancer, kidney cancer, urinary epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer, placental villi Cancer, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, digestive tract stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, prickle cell cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma, anti-tumor agent.
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